Phenyl xanthene dyes

ABSTRACT

Fluorescent phenyl xanthene dyes are described that comprise any fluorescein, rhodamine or rhodol comprising a particular C9 phenyl ring. One or both of the ortho groups on the lower C9 phenyl ring is ortho substituted with a group selected from alkyl, heteroalkyl, alkoxy, halo, haloalkyl, amino, mercapto, alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitroso, nitro, azido, sulfeno, sulfinyl, and sulfino. In one embodiment, halo and/or hydroxy groups are used. Optimal dyes contain a lower C9 phenyl ring in which both ortho groups are the same and the lower ring exhibits some form a symmetry relative to an imaginary axis running from the phenyl rings point of attachment to the remainder of the xanthene dye through a point para to the point of attachment. The phenyl xanthene dyes may be activated. Furthermore, the phenyl xanthene dyes may be conjugated to one or more substances including other dyes. The phenyl xanthene dyes are useful for a number of purposes, including labels for use in automated DNA sequencing as well the formation of fluorescent “bar codes” for polymeric particles used in the multiplexed analysis of analytes.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims benefit to U.S. provisional patentapplicaton No. 60/469,031, filed May 9, 2003, entitled “Phenyl XantheneDyes,” the entire disclosure of which is incorporated herein byreference.

2. BACKGROUND OF THE INVENTION

[0002] 2.1. Field of the Invention

[0003] Fluorescent phenyl xanthene dyes are described herein. Morespecifically, fluorescent phenyl rhodamines, phenyl fluoresceins andphenyl rhodols are described herein

[0004] 2.2 Description of Related Art

[0005] Dyes, including various fluorescein, rhodamine and rhodol dyes,are known. However, there is an ever present need to develop improvedfluorescent dyes, especially dyes that exhibit enhanced fluorescence andenhanced stability. In addition, there is a need to develop dyes thatcan be employed, if desired, in polymeric beads or particles. These andother needs are met by the various dyes described herein.

3. SUMMARY OF THE INVENTION

[0006] Phenyl xanthene dyes are described herein that exhibit usefulfluorescent properties. The phenyl xanthene dyes comprise anyfluorescein, rhodol or rhodamine ring system where the phenylsubstituent at the 9-carbon (“the C9 phenyl ring”) is a specific type ofphenyl ring. For the purposes of this description, fluoresceins, rhodolsand rhodamines are numbered in the following manner:

[0007] where A is either a hydroxyl or an amine group and B is a eitheran oxo or an imminium group. The C9 phenyl ring on the phenyl xanthenedye, whether substituted or unsubstituted, may be referred to as the“lower ring.” The remainder of the molecule may be referred to as the“upper ring system.”

[0008] The C9 phenyl ring is substituted at one or both of carbons C11or C15 with a group selected from alkyl, heteroalkyl, alkoxy, halo,haloalkyl, amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato,nitro, and sulfinyl. When both the C11 and C15 carbons are substituted,the substitutents may be the same or different. Thus, in one embodiment,the phenyl xanthene dyes comprise any fluorescein, rhodol, or rhodaminethat comprises a C9 phenyl ring comprising the following structure:

[0009] where at least one of R¹¹ or R¹⁵ is selected from alkyl,heteroalkyl, alkoxy, halo, haloalkyl, amino, alkylthio, cyano, isocyano,cyanato, mercaptocyanato, nitro, and sulfinyl.

[0010] The remaining carbons on the C9 phenyl ring can, independently ofone another, be unsubstituted or substituted with any group having nomore than 40 atoms and typically no more than 25 atoms. Illustrativesubstituent groups that can be positioned at carbons C12, C13 and/or C14include alkyl, heteroalkyl, aryl, heteroaryl, alkoxy, halo, haloalkyl,amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro,sulfinyl, sulfonyl, sulfonamide, carboxyl, and carboxyamide.Accordingly, in another embodiment, at least one of R¹¹ or R¹⁵ issubstituted as described above and the remainder of R¹¹, R¹², R¹³, R¹⁴and R¹⁵ are, independently of one another, selected from hydrogen,alkyl, heteroalkyl, aryl, heteroaryl, alkoxy, halo, haloalkyl, amino,alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro, sulfinyl,sulfonyl, sulfonamide, carboxyl, and carboxyamide.

[0011] It has been discovered that phenyl xanthene dyes, including a C9phenyl that is substituted with halo, haloalkyl, alkoxy and/or nitrilesubstituents, exhibit especially good fluorescent propertiesparticularly when placed at the C11 and/or C15 carbons. Accordingly, inanother embodiment, at least one of R¹¹ and R¹⁵ is selected from analkoxy, halo, haloalkyl and/or nitrile. In yet another embodiment, R¹¹and R¹⁵ are each, independently of one another, an alkoxy, halo,haloalkyl and/or nitrile.

[0012] In still another embodiment, at least one of R¹¹ and R¹⁵ isselected from an alkoxy, halo and/or haloalkyl and the remainder of R¹¹,R¹², R¹³, R¹⁴ and R¹⁵ are, independently of one another, selected fromhydrogen, alkoxy, halo and/or haloalkyl. In another embodiment, R¹¹ andR¹⁵ are each, independently of one another, an alkoxy, halo and/orhaloalkyl and the remainder of R¹², R¹³ and R¹⁴ are, independently ofone another, selected from hydrogen, alkoxy, halo and/or haloalkyl. Anyalkoxy and/or halo and/or haloalkyl groups present on the lower phenylring may be the same or different. However, in one embodiment, anyalkoxy and/or halo and/or haloalkyl groups present on the lower phenylring is identical to any other alkoxy and/or halo and/or haloalkylgroups present on the phenyl ring. Furthermore, in one embodiment, thelower phenyl ring is only substituted with hydrogen, alkoxy, halo and/orhaloalkyl groups.

[0013] Especially suitable alkoxy groups include (C1 to C20) oxyalkyls,particularly methoxy. In one embodiment, the phenyl ring is onlysubstituted with hydrogen and identical alkoxy groups. In one embodimentat least two groups on the phenyl ring are alkoxy. In another embodimentat least three groups on the phenyl ring are alkoxy. In anotherembodiment at least four groups on the phenyl ring are alkoxy. Inanother embodiment all of the groups on the phenyl ring are alkoxy.

[0014] Especially suitable halos include chloro and fluoro groups. Inone embodiment, the phenyl ring is only substituted with hydrogen andidentical halo groups, such as fluoro or chloro. In one embodiment atleast two groups on the phenyl ring are halo. In another embodiment atleast three groups on the phenyl ring are halo. In another embodiment atleast four groups on the phenyl ring are halo. In another embodiment allof the groups on the phenyl ring are halo.

[0015] Especially suitable haloalkyls include —CF3. Accordingly, in oneembodiment, the phenyl ring is only substituted with hydrogen andhaloalkyl groups such as —CF3 groups. In one embodiment at least twogroups on the phenyl ring are haloalkyl. In another embodimet at leastthree groups on the phenyl ring are haloalkyl. In another embodiment atleast four groups on the phenyl ring are haloalkyl. In a anotherembodiment all of the groups on the phenyl ring are haloalkyl.

[0016] Embodiments where the C9 phenyl ring is substituted at both theC11 and C15 carbons also exhibit especially good fluorescent properties.Accordingly, in one embodiment, R¹¹ and R¹⁵ are each, independently ofone another, selected from alkyl, heteroalkyl, alkoxy, halo, haloalkyl,amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro, andsulfinyl. The remaining carbons on the phenyl need not be substitutedand, if substituted, the substituents may, independently, be the same ordifferent when compared to R¹¹ and/or R¹⁵.

[0017] Embodiments where the C9 phenyl ring is identically substitutedat both carbons ortho to the point of the phenyl ring's attachment tothe remainder of the phenyl xanthene dye also exhibit desirablefluorescent properties. Accordingly, in another embodiment, R¹¹ and R¹⁵are identical. Once again, the remaining carbons on the phenyl need notbe substituted and, if substituted, the substituents may, independently,be the same or different when compared to R¹¹ and R¹⁵. In oneembodiment, any substituents on the lower phenyl ring are identical.

[0018] Symmetry appears to be an important factor in selecting optimalC9 phenyl rings. In this regard, the symmetry is relative to animaginary axis running from the lower phenyl ring's point of attachmentto the remainder of the phenyl xanthene dye (i.e., the 10-carbon)through a point para to the attachment (i.e., the 13-carbon).Accordingly, in one embodiment, R¹¹ and R¹⁵ are identical and theremainder of R¹², R¹³ and R¹⁴ are, identically, either hydrogen or asubstituent different from R¹¹ and R¹⁵. In another embodiment R¹¹, R³,and R¹⁵ are identical and the remainder of R¹², R¹³, and R¹⁴ are,identically, either hydrogen or a substituent different from R¹¹ , R¹³and R¹⁵. In yet another embodiment, R¹¹, R¹², R¹⁴ and R¹⁵ are identicaland R¹³ is either hydrogen or a substituent different from R¹¹ , R¹²,R¹⁴ and R¹⁵. In still another embodiment, R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ areall identical. Optimal lower phenyl rings include those where the ringexhibits one of the aforementioned symmetries and R¹¹ and R¹⁵ areselected from the same alkoxy and/or halo and/or haloalkyl groups.

[0019] The C9 phenyl ring departs from known C9 phenyl rings in phenylxanthene dyes in many ways. For example, as evident from the patentliterature, it is conventional wisdom to substitute the ortho phenylposition with a carboxyl or sulfonyl group, or some derivative thereof,such as an ester, amide, acid halide or salt. See, e.g., U.S. Pat. No.6,248,884, U.S. Pat. No. 6,229,055, U.S. Pat. No. 5,936,087, U.S. Pat.No. 5,847,162, U.S. Pat. No. 5,840,999, U.S. Pat. No. 5,750,409, U.S.Pat. No. 5,654,442, U.S. Pat. No. 5,442,045, U.S. Pat. No. 5,410,053,U.S. Pat. No. 5,366,860, U.S. Pat. No. 5,231,191, U.S. Pat. No.5,188,934, U.S. Pat. No. 5,066,580, U.S. Pat. No. 4,481,136 and U.S.Pat. No. 4,439,356. However, the instant C9 phenyl rings do not containthe aforementioned ortho carboxyl, ortho sulfonyl, or an ester, amide,acid halide, or salt thereof.

[0020] The C9 phenyl ring can be connected to any fluorescein, rhodol orrhodamine type upper ring system. Rhodamines are phenyl xanthenes thatadditionally comprise an exocyclic amine group and an exocyclic imminiumgroup. Rhodols are phenyl xanthenes that additionally comprise anexocyclic amine group and an exocyclic oxo group. Fluoresceins arephenyl xanthenes that additionally comprise an exocyclic hydroxyl groupand an exocyclic oxo group. The phenyl xanthene dyes described canemploy any fluorescein, rhodol and rhodamine type upper ring system aslong as the C9 phenyl attached thereto is as described herein.Accordingly, in one embodiment, the phenyl xanthene dye comprises afluorescein type upper ring system. In another embodiment, the phenylxanthene dye comprises a rhodol type upper ring system. In anotherembodiment, the phenyl xanthene dye comprises a rhodamine type upperring system.

[0021] Suitable fluorescein, rhodamine and rhodol type upper ringsystems are provided, for example, in U.S. Pat. No. 6,248,884, U.S. Pat.No. 6,229,055, U.S. Pat. No. 5,936,087, U.S. Pat. No. 5,847,162, U.S.Pat. No. 5,840,999, U.S. Pat. No. 5,750,409, U.S. Pat. No. 5,654,442,U.S. Pat. No. 5,442,045, U.S. Pat. No. 5,410,053, U.S. Pat. No.5,366,860, U.S. Pat. No. 5,231,191, U.S. Pat. No. 5,188,934, U.S. Pat.No. 5,066,580, U.S. Pat. No. 4,481,136 and U.S. Pat. No. 4,439,356, allof which relate to phenyl xanthenes and all of which are herebyincorporated by reference. However, the upper ring system is not limitedto the structures described in these patents. As stated, anyfluorescein, rhodol or rhodamine type upper ring system can be employedas long as it is attached to the C9 phenyl ring described herein.

[0022] Furthermore, as known in the art, phenyl xanthene dyes can beextended to include a 3,4- and/or a 5,6-benzo substituent (see, e.g.,U.S. Pat. No. 6,248,884, U.S. Pat. No. 5,750,409 and U.S. Pat. No.5,066,580). In “extended” fluoresceins, rhodols and rhodamines, theexocyclic amine or hydroxyl group and/or the exocyclic imminium or oxogroup are attached to any present 3,4- and/or 5,6-benzo substituents.These “extended” fluorescein, rhodol and rhodamine rings can also beemployed in the invention as long as they comprise the C9 phenyldescribed herein. Accordingly, the “fluorescein,” “rhodol” and“rhodamine” as used herein embrace extended structures.

[0023] In one embodiment, the phenyl xanthene dyes not only contain thenew lower phenyl ring but also contain sufficient lipophilic groups tomake the phenyl xanthenes lipid soluble. This is especially beneficialwhen the phenyl xanthenes are used, for example, to imbibe hydrophobicpolymeric particles that are useful in aqueous assays. Non-limitingexamples of such polymeric particles include crosslinked anduncrosslinked polystyrene particles and styrene-(meth) acrylic acidcopolymers. As evident to one of ordinary skill in the art, an unlimitedvariety of particles for use in assays are commercially available,including particles that are functionalized and/or paramagnetic and/orconjugated with one or more biological reagents. In such embodiments,the degree of lipid solubility required for the phenyl xanthene dyenecessarily varies as a function of the polymer utilized, the aqueoussolvent or solvent system employed in the assay in which the polymericparticle is to be used, and the conditions (e.g., time, temperature,pressure, pH, etc.) under which the assay is run. Suitable degrees oflipid solubility are easily determined by methods known in the art. Forexample, suitable lipid solubility can be determined by a partition testwherein a known quantity of dye in organic solvent is combined with theaqueous solvent or solvent system used in the assay. If a partitionresults and, under the conditions used in the assay, there is noappreciable crossing by the dye into the solvent or solvent system, thenthe dye is sufficiently lipid soluble. Put another way, the lipidsoluble phenyl xanthene dye should be sufficiently lipid soluble suchthat it is capable of being imbibed into the polymer when dissolved inan organic solvent or solvent system and, when the dyed polymer issubjected to the aqueous conditions of the assay, the dye should resistleaching out of the polymer to any degree that significantly impacts thefluorescent signature of the dye imbibed polymer or the results of theassay.

[0024] In those embodiments where the phenyl xanthene dyes are lipidsoluble rhodamines, one or both of the exocyclic amine and exocyclicimminium nitrogens are often substituted with one or more lipophilicgroups designed to impart to the rhodamine lipophilic characteristics orproperties. Thus, useful dyes include rhodamines that comprise the lowerphenyl ring described above and also comprise one or two lipophilicsubstituents at the exocyclic amine nitrogen and/or one or twolipophilic substituents at the exocyclic imminium nitrogen. In oneembodiment, both the exocyclic amine nitrogen and the exocyclic imminiumnitrogen are substituted with a lipophilic group. In another embodiment,the exocyclic amine nitrogen and the exocyclic imminium nitrogen areboth substituted with two lipophilic groups. The lipophilic groups,whether attached to the same or different exocyclic nitrogen, may be thesame or different. In one embodiment, the lipophilic groups on theexocyclic nitrogens are the same.

[0025] In those embodiments where the phenyl xanthene dyes are lipidsoluble rhodols, the exocyclic amine nitrogen is often substituted withone or more lipophilic groups designed to impart to the rhodollipophilic characteristics or properties. Thus, useful dyes includerhodols that comprise the C9 phenyl described herein and also compriseone or two lipophilic substituents at the exocyclic amine nitrogen. Inone embodiment, the exocyclic amine nitrogen is substituted with onelipophilic group. In another embodiment, the exocyclic amine nitrogen issubstituted with two lipophilic groups. If there are two lipophilicgroups on the exocyclic amine nitrogen, the lipophilic groups may besame or different. In one embodiment, there are two lipophilic groups onthe exocyclic amine nitrogen that are the same.

[0026] Lipid-soluble phenyl xanthene dyes may include lipophilicsubstituents at other positions as well. It is the net effect of thelipophilic substituents that determines whether the phenyl xanthene dyeis lipid soluble. This is especially true for fluoresceins which have noexocyclic amine or imminium nitrogens.

[0027] Lipophilic substituents are groups that impart the resultantphenyl xanthene dye with lipophilic characteristics or properties asdenoted above. The nature of each lipophilic substituent is notcritical, as long as the resultant phenyl xanthene dye is lipid soluble.Non-limiting examples of suitable lipophilic substituents includeunsubstituted (C4-C20) alkyls, (C5-C40) aryls, and (C6-C40) arylalkyls.Depending on the number of methylene and methine units in the lipophilicsubstituent, the lipophilic substituent may also include pendant orinternal polar or hydrophilic groups. For example, a lipophilicsubstituent may include one or more internal heteroatoms, such as one ormore internal O, S, N or NH groups. As another example, a lipophilicsubstituent may include one or more pendant polar or hydrophilicsubstituents, such as one or more pendant halogen, —OH, —SH, —NH₂,—C(O)OH, —C(O)NH₂ or other polar or hydrophilic groups. Thus, lipophilicsubstituents may also include substituted (C4-C20) alkyl, substituted(C5-C40) aryls and substituted (C6-C40) arylalkyls, as well assubstituted and unsubstituted (C4-C20) heteroalkyl, substituted andunsubstituted (C5-C40) heteroaryls and substituted and unsubstituted(C6-C40) arylalkyls. The number of internal or pendant polar orhydrophilic groups that may be included in a lipophilic substituent willdepend upon, among other factors, the number of methylene or methinegroups included in the lipophilic substituent and the number oflipophilic substituents on the phenyl xanthene dye. The nature andnumber of lipophilic groups necessary to make a phenyl xanthene lipidsoluble can vary from molecule to molecule, and will be apparent tothose of skill in the art.

[0028] Oftentimes, it is desirable to attach fluorescent dyes such asthe phenyl xanthene dyes described herein to substances such as solidsupports, particles, and biological and non-biological molecules (e.g.,drugs, amino acids, peptides, polypeptides, proteins, nucleosides,nucleotides, oligonucleotides, polynucleotides, carbohydrates, etc.)Thus, in one embodiment, the various phenyl xanthene dyes describedherein include one or more moieties suitable for such attachment. Suchmoieties are expressed by the formula —S-LG where S is a direct bond ora spacing moiety and LG is a linking group capable of forming a linkagewith the substance to be conjugated. The linking group LG may be anymoiety capable of forming the linkage, which may be covalent ornon-covalent. For example, the linking group may be one member of a pairof specific binding molecules that non-covalently bind one another, suchas biotin and avidin/streptavidin. Thus, in one embodiment, the linkinggroup is biotin. Alternatively, the linking group may be a functionalgroup capable of forming a covalent linkage with a “complementary”functional group, such as an electrophilic (or nucleophilic) group whichis capable of forming a covalent linkage with a complementarynucleophilic (or electrophilic) group, although other groups may be useddepending on the desired linking chemistry, as is well known in the art.The linking group may be attached directly to the phenyl xanthene dye orit may be spaced away from the phenyl xanthene dye by way of spacingmoiety “S.” As will be appreciated by skilled artisans, the nature andcomposition of the spacing moiety is not critical and may depend uponthe particular application. The linking group, whether attached directlyor spaced away via spacing moiety “S,” may be attached to any availableposition of the phenyl xanthene dye. For example, the linking group maybe attached to any available position on the upper ring system or thelower ring. In one embodiment, the linking group —S-LG is attached tothe C2, C4, C5, or C7 position of the upper ring system. In anotherembodiment, the linking group —S-LG is attached to the C12, C13 or C14position of the lower ring.

[0029] Alternatively, the lipid-soluble phenyl xanthene dyes may belinked to a conjugated substance. In this embodiment, at least onesubstituent on the phenyl xanthene dye is —S¹-LK—S²—CS, where S¹ and S²are, independently of one another, a direct bond or a spacing moiety, LKrepresents a linkage, which may be a bond or another type of linkage,and CS is a conjugated substance. Non-limiting examples of substancesthat can be conjugated include glass substrates, metal substrates,polymeric substrates, biomolecules, haptens, drugs, poisons, vitamins,antigens, and pathogens. Once again, the linker will vary depending theidentity of the conjugated substance.

[0030] Similarly, the phenyl xanthene dye may be part of an energytransfer (“ET”) network comprising, for example, from two to four dyescovalently attached to one another that transfer energy to generate alonger Stoke's shift. In other words, the phenyl xanthene dye may bepart of series of dyes that are covalently attached to one another. Oneexample of an ET network would be a fluorescence resonance energytransfer (“FRET”) dye. In this embodiment, at least one substituent onthe phenyl xanthene dye is —S¹-LK-S²-D, where S¹ and S² are,independently of one another, a direct bond or a spacing moiety, LKrepresents a linkage, which may be a bond or another type of linkage,and D is a dye. Linkages for covalently attaching phenyl xanthene dyesto other dyes are known in the art, as are suitable locations forattachment to the phenyl xanthene dyes (see, e.g., U.S. Pat. Nos.5,800,996 and 5,863,727). In one embodiment, each dye in the energytransfer network is within 5 to 100 Å of the neighboring dye or dyes inthe network to which it is covalently attached. In such embodiments, thephenyl xanthene dye can be the donor, acceptor, or an intermediate dyein the network.

[0031] In a more particular embodiment, the phenyl xanthene dyes are anyfluorescent dye that comprises one of the following “core structures:”

[0032] where A is —OH or NR^(3′)R^(3″), where B is a ═O or═N^(⊕)R^(6′)R^(6″), where R¹¹ and R¹⁵ are, independently of one another,selected from alkyl, heteroalkyl, alkoxy, halo, haloalkyl, amino,alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro, andsulfinyl, and the remainder of R¹, R², R^(2′), R^(3′), R^(3″), R⁴,R^(4′), R⁵, R^(5′), R^(6′), R^(6″), R⁷, R^(7′), R⁸, R¹², R¹³, and R¹⁴are, independently of one another, selected from hydrogen and asubstituent having no more than 40 atoms, and typically no more than 25atoms. In one embodiment, the phenyl xanthene dye is lipid soluble. Inanother embodiment, one or more of the remainder of R¹, R², R^(2′),R^(3′), R^(3″), R⁴, R^(4′), R⁵, R^(5′), R^(6′), R^(6″), R⁷, R^(7′), R⁸,R¹², R¹³, and R¹⁴ may be —S-LG where S is a direct bond or a spacingmoiety and LG is a linking group. In another embodiment one or more ofthe remainder of R¹, R², R^(2′), R^(3′), R^(3″), R⁴ , R^(4′), R⁵,R^(5′), R^(6′), R^(6″), R⁷, R^(7′), R⁸, R¹², R¹³, and R¹⁴ may be—S¹-LK-S²—CS, where S¹ and S² are each, independently of one another, adirect bond or a spacing moiety, LK is a linkage, and CS is a conjugatedsubstance.

[0033] The phenyl xanthene dyes are useful in any commonly knownapplication for dyes. For example the dyes are useful as fluorescentlabels for automated DNA sequencing, oligonucleotide hybridizationmethods, detection of polymerase-chain reaction products, immunoassays,and the like. In addition, the dyes may be imbibed into polymericparticles for use in the standardization of fluorescence-basedinstrumentation, as a biological tracer, and in the detection andanalysis of biomolecules. In these latter applications, it is oftendesirable for the dyes to be lipid soluble as previously discussed.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 illustrates the synthesis of phenyl xanthene dyes;

[0035]FIGS. 2A and 2B illustrate the synthesis of extended phenylxanthene dyes;

[0036]FIG. 3 illustrates the synthesis of fluoresceins;

[0037]FIG. 4 illustrates the synthesis of extended fluoresceins; and

[0038]FIG. 5 illustrates the synthesis of a rhodol.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] 5.1 Numbering System

[0040] For the purposes of the present application, carbon atoms inphenyl xanthenes such as fluoresceins, rhodols and rhodamines, orextended versions thereof, are numbered in the following manner:

[0041] where A is either a hydroxyl (—OH) or an amine (—NH₂) and B iseither an oxo (═O) or an imminium (═NH₂ ^(⊕)).

[0042] 5.2 Definitions

[0043] As used herein, the following terms are intended to have thefollowing meanings:

[0044] “Phenyl Xanthene Dye,” as used herein, refers to any dye thatcomprises a xanthene ring or an extended xanthene ring that issubstituted with a C9 phenyl group, an exocyclic amine or hydroxyl groupand an excyclic imminium or oxo group, as shown in formulae (1), (2) and(3) above. As known in the art, various substitutions may be made forthe hydrogens on any of the 1-, 2-, 2′-, 4-, 4′-, 5′-, 5-, 7′-, 7-, 8-,11-, 12-, 13-, 14-, and 15-carbons, as well as any hydrogens on anyexocyclic amine or exocyclic imminium present. Substitutions can beindependently selected from any of a wide variety of the same ordifferent groups known in the art including, but not limited to, —X,—R^(S), —OR^(S), —SR^(S), —NR^(S)R^(S), perhalo, (C1-20) alkyl, —CX₃,—CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, —N₃, —S(O)₂O⁻, —S(O)₂OH,—S(O)₂R^(S), —C(O)R^(S), —C(O)X, —C(S)R^(S), —C(S)X, —C(O)OR^(S),—C(S)OR^(S), —C(O)SR^(S), —C(S)SR^(S), —C(O)NR^(S)R^(S),—C(S)NR^(S)R^(S) and —C(NR^(S))NR^(S)R^(S), where each X isindependently a halogen (e.g., fluoride or chloride), and each R^(S) isindependently hydrogen, (C1-C20) alkyl or heteroalkyl, (C5-C20) aryl orheteroaryl, and (C6-C40) arylalkyl or heteroarylalkyl. Any of theaforementioned substituents can, in turn, be further substituted withone or more of the same or different substituents.

[0045] Moreover, the 1- and 2-substituents or the 2- and2′-substituents, and/or the 7′ and 7 substituents or the 7- and8-substituents, can be taken together to form substituted orunsubstituted (C5-C20) benzo, naptho or polycyclic aryleno bridges. Thebridges may, in turn, be further substituted, for example, with any ofthe substituents R^(S) above.

[0046] When A is an amine and/or B is an imminium, the exocyclicnitrogen or nitrogens can be included in 5 or 6 membered rings involvingthe nitrogen atom and an adjacent carbon atom on the xanthene dye. Therings may, in turn, be further substituted, for example, with any of thesubstituents R^(S) above.

[0047] “Rhodamine,” as used herein, is a specific type of phenylxanthene dye. Rhodamines embrace any substituted or unsubstituted dyethat comprises one of formulae (1), (2) and (3) above, where A is asubstituted or unsubstituted amine group and B is a substituted orunsubstituted imminium group. Examples of the various substitutions thatmay be made for hydrogens at the 1-, 2-, 2′-, 4-, 4′-, 5′-, 5-, 7′-, 7-,and 8-carbons, as well as hydrogens at the amine and imminium nitrogensare illustrated, for example, in U.S. Pat. No. 6,372,907, U.S. Pat. No.6,248,884, U.S. Pat. No. 5,936,087, U.S. Pat. No. 5,847,162, U.S. Pat.No. 5,840,999, U.S. Pat. No. 5,750,409, U.S. Pat. No. 5,410,053, U.S.Pat. No. 5,366,860, and U.S. Pat. No. 5,231,191.

[0048] “Rhodol,” as used herein, is another specific type of phenylxanthene dye. Rhodols embrace any substituted or unsubstituted dye thatcomprises one of formulae (1), (2) and (3) above, where A is asubstituted or unsubstituted amine group and B is an oxo group. Examplesof the various substitutions that may be made for hydrogens at the 1-,2-, 2′-, 4-, 4′-, 5′-, 5-, 7′-, 7-, and 8-carbons, as well as hydrogensat the amine nitrogen are illustrated, for example, in U.S. Pat. No.6,372,907, U.S. Pat. No. 6,229,055, U.S. Pat. No. 6,008,379, U.S. Pat.No. 5,840,999, and U.S. Pat. No. 5,442,045.

[0049] “Fluorescein,” as used herein, is another specific type of phenylxanthene dye. Fluoresceins embrace any substituted or unsubstituted dyethat comprises one of formulae (1), (2) and (3) above, where A is ahydroxyl group and B is an oxo group. Examples of the varioussubstitutions that may be made for hydrogens at the 1-, 2-, 2′-, 4-,4′-, 5′-, 5-, 7′-, 7-, and 8-carbons are illustrated, for example, inU.S. Pat. No. 6,229,055, U.S. Pat. No. 5,840,999, U.S. Pat. No.5,654,442, U.S. Pat. No. 5,750,409, U.S. Pat. No. 5,188,934, U.S. Pat.No. 5,066,580, U.S. Pat. No. 4,481,136, and U.S. Pat. No. 4,439,356.

[0050] “Fluorescent Dye” or “Fluorescer” or “Fluorochrome” or“Fluorophore” as used interchangeably herein refer to molecules thatabsorb electromagnetic radiation at one wavelength and emitelectromagnetic radiation at another wavelength in passing from a higherto a lower electronic state.

[0051] “Carboxyl” as used herein, is defined to include not only thecarboxyl group (—COOH or —CO₂H) but also carboxylate radicals (—CO₂ ⁻).

[0052] “Sulfonyl,” as used herein, is defined to include not only thesulfonyl group (—SO₂OH or —SO₃H), but also sulfonate radicals (—SO₃ ⁻).

[0053] “Biomolecule” as used herein refers to a molecule of a typetypically found in a biological system, whether such molecule isnaturally occurring or the result of some external disturbance of thesystem (e.g., a disease, poisoning, genetic manipulation, etc.), as wellas synthetic analogs and derivatives thereof. Non-limiting examples ofbiomolecules include amino acids (naturally occurring or synthetic),peptides, polypeptides, glycosylated and unglycosylated proteins (e.g.,polyclonal and monoclonal antibodies, receptors, interferons, enzymes,etc.), nucleosides, nucleotides, oligonucleotides (e.g., DNA, RNA, PNAoligos), polynucleotides (e.g., DNA, cDNA, RNA, etc.), carbohydrates,hormones, haptens, steroids, toxins, etc. Biomolecules may be isolatedfrom natural sources, or they may be synthetic.

[0054] “Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C1-C6 means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like. Wherespecific levels of saturation are intended, the nomenclature “alkanyl,”“alkenyl” and/or “alkynyl” is used, as defined below. In preferredembodiments, the alkyl groups are (C1-C20) alkyl.

[0055] “Heteroalkyl,” by itself or as part of another substituent refersto an alkyl in which one or more of the carbon atoms are eachindependently replaced with the same or different heteroatoms orheteroatomic groups. Typical heteroatoms and/or heteroatomic groupswhich can replace the carbon atoms include, but are not limited to, —O—,—S—, —S—O—, —NR^(m)—, —PH—, —S(O)—, —S(O)₂—, —S(O) NR^(m)—,—S(O)₂NR^(m)—, and the like, including combinations thereof, where eachR^(m) is independently hydrogen or (C1-C6) alkyl.

[0056] “Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In preferred embodiments, the aryl groupis (C5-C15) aryl, with (C5-C10) being even more preferred. Particularlypreferred aryls are phenyl and naphthyl.

[0057] “Arylaryl” by itself or as part of another substituent refers toa monovalent hydrocarbon group derived by the removal of one hydrogenatom from a single carbon atom of a ring system in which two or moreidentical or non-identical parent aromatic ring systems are joineddirectly together by a single bond, where the number of such direct ringjunctions is one less than the number of parent aromatic ring systemsinvolved. Typical arylaryl groups include, but are not limited to,biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, andthe like. Where the number of carbon atoms in an arylaryl group arespecified, the numbers refer to the carbon atoms comprising each parentaromatic ring. For example, (C5-C15) arylaryl is an arylaryl group inwhich each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl,triphenyl, binaphthyl, phenylnaphthyl, etc. Preferably, each parentaromatic ring system of an arylaryl group is independently a (C5-C15)aromatic, more preferably a (C5-C10) aromatic. Also preferred arearylaryl groups in which all of the parent aromatic ring systems areidentical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.

[0058] “Arylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In preferred embodiments, the arylalkyl group is(C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C1-C6) and the aryl moiety is (C5-C15). Inparticularly preferred embodiments the arylalkyl group is (C6-C13),e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is(C1-C3) and the aryl moiety is (C5-C10).

[0059] “Heteroaryl” by itself or as part of another substituent refersto a monovalent heteroaromatic group having the stated number of ringatoms (e.g., “5-14 membered” means from 5 to 14 ring atoms) derived bythe removal of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In preferredembodiments, the heteroaryl group is a 5-14 membered heteroaryl, with5-10 membered heteroaryl being particularly preferred.

[0060] “Heteroaryl-Heteroaryl” by itself or as part of anothersubstituent refers to a monovalent heteroaromatic group derived by theremoval of one hydrogen atom from a single atom of a ring system inwhich two or more identical or non-identical parent heteroaromatic ringsystems are joined directly together by a single bond, where the numberof such direct ring junctions is one less than the number of parentheteroaromatic ring systems involved. Typical heteroaryl-heteroarylgroups include, but are not limited to, bipyridyl, tripyridyl,pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified,the numbers refer to the number of atoms comprising each parentheteroaromatic ring systems. For example, 5-15 memberedheteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which eachparent heteroaromatic ring system comprises from 5 to 15 atoms, e.g.,bipyridyl, tripuridyl, etc. Preferably, each parent heteroaromatic ringsystem is independently a 5-15 membered heteroaromatic, more preferablya 5-10 membered heteroaromatic. Also preferred are heteroaryl-heteroarylgroups in which all of the parent heteroaromatic ring systems areidentical.

[0061] “Heteroarylalkyl” by itself or as part of another substituentrefers to an acyclic alkyl group in which one of the hydrogen atomsbonded to a carbon atom, typically a terminal or sp³ carbon atom, isreplaced with a heteroaryl group. Where specific alkyl moieties areintended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/orheteroarylalkynyl is used. In preferred embodiments, the heteroarylalkylgroup is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and theheteroaryl moiety is a 5-15-membered heteroaryl. In particularlypreferred embodiments, the heteroarylalkyl is a 6-13 memberedheteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3)alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.

[0062] “Halogen” or “Halo” by themselves or as part of anothersubstituent, unless otherwise stated, refer to fluoro, chloro, bromo andiodo.

[0063] “Haloalkyl” by itself or as part of another substituent refers toan alkyl group in which one or more of the hydrogen atoms is replacedwith a halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C1-C2) haloalkyl” includes 1-fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0064] The above-defined groups may include prefixes and/or suffixesthat are commonly used in the art to create additional well-Recognizedsubstituent groups. As examples, “allyloxy” or “alkoxy” refers to agroup of the formula —OR^(n), “alkylamine” refers to a group of theformula —NHR^(n) and “dialkylamine” refers to a group of the formula—NR^(n)R^(n), where each R^(n) is independently an alkyl. As anotherexample, “haloalkoxy” or “haloalkyloxy” refers to a group of the formulaOR^(p), where R^(p) is a haloalkyl.

[0065] 5.3 Phenyl Xanthene Dyes

[0066] The phenyl xanthene dyes include any fluorescein, rhodol orrhodamine that comprises the C9 phenyl ring described herein.Accordingly, any rhodamine, rhodol and fluorescein type upper ringsystem may be employed. Suitable rhodamine type upper ring systems aredescribed in U.S. Pat. No. 6,248,884, U.S. Pat. No. 6,229,055, U.S. Pat.No. 5,936,087, U.S. Pat. No. 5,847,162, U.S. Pat. No. 5,840,999, U.S.Pat. No. 5,750,409, U.S. Pat. No. 5,654,442, U.S. Pat. No. 5,442,045,U.S. Pat. No. 5,410,053, U.S. Pat. No. 5,366,860, U.S. Pat. No.5,231,191, U.S. Pat. No. 5,188,934, U.S. Pat. No. 5,066,580, U.S. Pat.No. 4,481,136 and U.S. Pat. No. 4,439,356, all of which are incorporatedherein by reference. However, the upper ring systems are not limited bythese patents. As stated, any fluorescein, rhodol or rhodamine coreupper ring system can be employed.

[0067] The C9 phenyl ring is substituted at one or both of carbons C11or C15 with a group selected from alkyl, heteroalkyl, alkoxy, halo,haloalkyl, amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato,nitro, and sulfinyl. When both the C11 and C15 carbons are substituted,the substitutents may be the same or different. Thus, in one embodiment,the phenyl xanthene dyes include any fluorescein, rhodol, or rhodamineupper ring system that is substituted at the C9 carbon with a phenylring comprising the following structure:

[0068] where at least one of R¹¹ or R¹⁵ is selected from alkyl,heteroalkyl, alkoxy, halo, haloalkyl, amino, alkylthio, cyano, isocyano,cyanato, mercaptocyanato, nitro, and sulfinyl.

[0069] The remaining carbons on the C9 phenyl ring can, independently ofone another, be unsubstituted or substituted with any group having nomore than 40 atoms and typically no more than 25 atoms. Illustrativesubstituent groups that can be positioned at carbons C12, C13 and/or C14include alkyl, heteroalkyl, aryl, heteroaryl, alkoxy, halo, haloalkyl,amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro,sulfinyl, sulfonyl, sulfonamide, carboxyl and carboxyamide. Accordingly,in another embodiment, at least one of R¹¹ or R¹⁵ is substituted asdescribed above and the remainder of R¹¹ , R¹², R¹³, R¹⁴ and R¹⁵ are,independently of one another, selected from hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, alkoxy, halo, haloalkyl, amino,alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro, sulfinyl,sulfonyl, sulfonamide, carboxyl and carboxyamide.

[0070] As long as at least one of R¹¹ and R¹⁵ is substituted asdescribed above, substituents at the remaining carbons in the C9 phenylring may be absent or present in any conceivable combination. This isillustrated by the following exemplary C9 phenyl structures, wherein oneor both of R¹¹ and R¹⁵ is R^(O) and the remaining carbons on the phenylare either unsubstituted or substituted with R^(S), wherein each R^(O),independently, is a group selected from alkyl, heteroalkyl, alkoxy,halo, haloalkyl, amino, alkylthio, cyano, isocyano, cyanato,mercaptocyanato, nitro, and sulfinyl, and each R^(S), independently, isany substituent having up to 40 atoms.

[0071] It has been discovered that xanthene dyes that include a C9phenyl substituted with halo, haloalkyl, alkoxy and/or nitrilesubstituents exhibit especially good fluorescent properties,particularly when the subsituents are placed at the C11 and/or C15carbons. Accordingly, in another embodiment, at least one of R¹¹ and R¹⁵is selected from an alkoxy, halo, haloalkyl and/or nitrile. In yetanother embodiment, R¹¹ and R¹⁵ are each, independently of one another,an alkoxy, halo, haloalkyl and/or nitrile.

[0072] In still another embodiment, at least one of R¹¹ l and R¹⁵ isselected from an alkoxy, halo and/or haloalkyl and the remainder of R¹¹,R¹², R¹³, R¹⁴ and R¹⁵ are, independently of one another, selected fromhydrogen, alkoxy, halo and/or haloalkyl. In another embodiment, R¹¹ andR¹⁵ are each, independently of one another, an alkoxy, halo and/orhaloalkyl and the remainder of R¹², R¹³ and R¹⁴ are, independently ofone another, selected from hydrogen, alkoxy, halo and/or haloalkyl. Anyalkoxy and/or halo and/or haloalkyl group present on the lower phenylring may be the same or different. However, in one embodiment, anyalkoxy and/or halo and/or halo alkyl group present on the lower phenylring is identical to any other alkoxy and/or halo and/or haloalkyl grouppresent on the phenyl ring. Furthermore, in one embodiment, the lowerphenyl ring is only substituted with hydrogen, alkoxy, halo and/orhaloalkyl groups.

[0073] Especially suitable alkoxy groups include (C1 to C20) oxyalkyls,particularly methoxy. In one embodiment, the phenyl ring is onlysubstituted with hydrogen and identical alkoxy groups. In one embodimentat least two groups on the phenyl ring are alkoxy. In another embodimentat least three groups on the phenyl ring are alkoxy. In anotherembodiment at least four groups on the phenyl ring are alkoxy. Inanother embodiment all of the groups on the phenyl ring are alkoxy.

[0074] Especially suitable halos include chloro and fluoro groups. Inone embodiment, the phenyl ring is only substituted with hydrogen andidentical halo groups, such as fluoro or chloro. In one embodiment atleast two groups on the phenyl ring are halo. In another embodiment atleast three groups on the phenyl ring are halo. In another embodiment atleast four groups on the phenyl ring are halo. In another embodiment allof the groups on the phenyl ring are halo.

[0075] Especially suitable haloalkyls include —CF3. Accordingly, in oneembodiment, the phenyl ring is only substituted with hydrogen andhaloalkyl groups such as —CF3 groups. In one embodiment at least twogroups on the pheny ring are haloalkyl. In another embodimet at leastthree groups on the phenyl ring are haloalkyl. In another embodiment atleast four groups on the phenyl ring are alkoxy. In another embodimentall of the groups on the phenyl ring are haloalkyl.

[0076] Embodiments where the C9 phenyl ring is substituted at both theC11 and C15 carbons also exhibit especially good fluorescent properties.Accordingly, in one embodiment, R¹¹ and R¹⁵ are each, independently ofone another, selected from alkyl, heteroalkyl, alkoxy, halo, haloalkyl,amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato, nitro, andsulfinyl. The remaining carbons on the phenyl need not be substitutedand, if substituted, the substituents may, independently, be the same ordifferent when compared to R¹¹ and/or R¹⁵.

[0077] Embodiments where the C9 phenyl ring is identically substitutedat both carbons ortho to the point of the phenyl ring's attachment tothe remainder of the xanthene dye also exhibit desirable fluorescentproperties. Accordingly, in another embodiment, R¹¹ and R¹⁵ areidentical. Once again, the remaining carbons on the phenyl need not besubstituted and, if substituted, the substituents may, independently, bethe same or different when compared to R¹¹ and R¹⁵. In one embodiment,any substituents on the lower phenyl ring are identical.

[0078] Symmetry appears to be an important factor in selecting optimalC9 phenyl rings. In this regard, the symmetry is relative to animaginary axis running from the lower phenyl ring's point of attachmentto the remainder of the xanthene dye (i.e., the 10-carbon) through apoint para to the attachment (i.e., the 13-carbon). Accordingly, in oneembodiment, R¹¹ and R¹⁵ are identical and the remainder of R¹², R¹³ andR¹⁴ are, identically, either hydrogen or a substituent different fromR¹¹ and R¹⁵. In another embodiment R¹¹, R¹³, and R¹⁵ are identical andthe remainder of R¹² and R¹⁴ are, identically, either hydrogen or asubstituent different from R¹¹, R¹³ and R¹⁵. In yet another embodiment,R¹¹, R¹², R¹⁴ and R¹⁵ are identical and R¹³ is either hydrogen or asubstituent different from R¹¹, R¹², R¹⁴ and R¹⁵. In still anotherembodiment, R¹¹ , R¹², R¹³, R¹⁴ and R¹⁵ are all identical. Optimal lowerphenyl rings include those where the ring exhibits one of theaforementioned symmetries and R¹¹ and R¹⁵ are selected from the samealkoxy and/or halo and/or haloalkyl group.

[0079] For the purposes of illustration, the following non-limitingexamples of symmetrical halo and alkoxy substituted lower phenyl ringsare provided, wherein X represents any halo group and R^(A) represents a(C1 to C20) alkyl:

[0080] As stated, the fluorescent phenyl xanthene dyes comprise anyfluorescein, rhodol or rhodamine that comprises the C9 phenyl ringdiscussed above. Illustrative phenyl xanthene dyes include dyes thatcomprise one of the following “core structures:”

[0081] where A is —OH or NR^(3′)R^(3″),

[0082] where B is a ═O or ═N^(⊕)R^(6′)R^(6″),

[0083] where R¹¹ and R¹⁵ are selected from alkyl, heteroalkyl, alkoxy,halo, haloalkyl, amino, alkylthio, cyano, isocyano, cyanato,mercaptocyanato, nitro, and sulfinyl,

[0084] and the remainder of R¹, R², R^(2′), R^(3′), R^(3″), R⁴, R^(4′),R⁵, R^(5′), R^(6′), R^(6″), R⁷, R^(7′), R⁸, R¹², R¹³, and R¹⁴ are,independently, selected from hydrogen and a substituent having no morethan 40 atoms, typically no more than 25 atoms.

[0085] In one embodiment the phenyl xanthene dyes are rhodamines,namely, when A is an amine group and B is an imminium group. In analternative embodiment the phenyl xanthene dyes are rhodols, namely,when A is an amine and B is a oxo group. In an alternative embodiment,the phenyl xanthene dyes are fluoresceins, namely, when A is a hydroxylgroup and B is a oxo group.

[0086] The signal emitted by the phenyl xanthene dyes can be tuned bythe selection of different substituents. Especially beneficialsubstituents for tuning the phenyl xanthene dyes include R¹¹, R¹², R¹³,R¹⁴ and R¹⁵, with R¹¹ and R¹⁵ being exceptionally effective for tuningthe dyes. The R², R^(2′), R⁴, R^(4′), R^(5′), R⁵, R^(7′) and R⁷substituents are also beneficial toward tuning the spectral propertiesof the dyes.

[0087] Usually, R¹ and R⁸ are not, simultaneously, pendant or fusedbenzo, naphtho or polycyclic aryleno rings. The simultaneous presence oftwo relatively rigid aromatic substituents immediately next to the9-carbon phenyl may generate steric hinderances.

[0088] As stated, symmetry can be an important factor in selectingoptimal C9 phenyl rings. Symmetry can also be a factor in selectingoptimal fluorescein, rhodol and rhodamine type upper ring systems, aswell as a factor in selecting optimal phenyl xanthene dyes overall.Accordingly, it is desirable, but not necessary, for the phenyl xanthenedyes to have identical R^(3′) and R^(6′) substituents (if present)and/or identical R^(3″) and R^(6″) substituents (if present) and/oridentical R⁴ and R⁵ substituents. Similarly, it is desirable, but notnecessary, for the dyes to have identical R¹ and R⁸ substituents and/oridentical R² and R⁷ substituents. Similarly, it is often desirable forR¹¹ and R¹⁵, as well as R¹² and R¹⁴′ to be identical. The presence ofone or more, and especially all, of these symmetries facilitates theproduction of a strong signal.

[0089] In one embodiment, the phenyl xanthene dyes comprise corestructure (I) and, additionally, the substituents therein are defined asfollows:

[0090] A is selected from —OH and —NR^(3′)R^(3″);

[0091] B is selected from ═O and ═NR^(⊕)R^(6′)R^(6″);

[0092] R¹ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R¹ may be taken together with R² toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups;

[0093] R² is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R² may be taken together with R¹ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, when A is —NR^(3′)R^(3″),R² may be taken together with R^(3′) to form a 5- or 6-membered ringwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups;

[0094] R^(3′), when present, is selected from hydrogen, (C1-C20) alkylor heteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3′) may be taken together with R² toform a 5- or 6-membered ring which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups;

[0095] R^(3″), when present, is selected from (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3″) may be taken together with R⁴ toform a 5- or 6-membered ring which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups;

[0096] R⁴ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, when B is —NR^(3′)R^(3″), R⁴ may betaken together with R^(3″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups;

[0097] R⁵ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) group or, alternatively, when B is —NR^(6′)R^(6″+), R⁵ may betaken together with R^(6″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups;

[0098] R^(6″), when present, is selected from (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups or, alternatively R^(6″) may be taken together with R⁵ toform a 5- or 6-membered ring which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups;

[0099] R^(6′), when present, is selected from hydrogen, (C1-C20) alkylor heteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(6′) may be taken together with R⁷ toform a 5- or 6-membered ring optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups;

[0100] R⁷ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or R^(b)groups, or, alternatively, R⁷ may be taken together with R⁸ to form partof a benzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, when B is —NR^(6′)R^(6″+), R⁷ may betaken together with R^(6′) to form a 5- or 6-membered ring optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups;

[0101] R⁸ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitable.R^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitable.R^(b) groups, or, alternatively, R⁸ together with R⁷ may form part of abenzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups;

[0102] R¹¹ and R¹⁵ are each, independently of one another, selected fromhalo, (C1-C20) alkyl, haloalkyl, —OR^(y), —SR^(y), —SOR^(y), —SO₂R^(y),and nitrile;

[0103] R¹², R¹³ and R¹⁴ are each, independently of one another, selectedfrom hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroarylalkyl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups;

[0104] R^(x) is selected from —NR^(c)R^(c), —OR^(d), —SR^(d), halo,haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and —OC(NH)NR^(c)R^(c);

[0105] R^(y) is selected from (C1-C20) alkyls or heteroalkyls optionallysubstituted with lipophilic substituents, (C5-C20) aryls or heteroarylsoptionally substituted with lipophilic substituents and (C2-C26)arylalkyl or heteroarylalkyls optionally substituted with lipophilicsubstituents;

[0106] R^(a) is selected from hydrogen, (C1-C8) alkyl or heteroalkyl,(C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl or heteroarylalkyl;

[0107] R^(b) is selected from —NR^(c)R^(c), ═O, —OR^(d), ═S, —SR^(d),═NR^(d), ═NOR^(d), halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂,═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and—OC(NH)NR^(c)R^(c);

[0108] each R^(c) is independently hydrogen or R^(d), or, alternatively,each R^(c) is taken together with the nitrogen atom to which it isbonded to form a 5 to 8-membered saturated or unsaturated ring which mayoptionally include one or more of the same or different additionalheteroatoms and which may optionally be substituted with one or more ofthe same or different R^(a) or R^(d) groups;

[0109] each R^(d) is independently R^(a) or R^(a) substituted with oneor more of the same or different R^(a) or R^(e) groups;

[0110] each R^(e) is selected from —NR^(a)R^(a), ═O, —OR^(a), ═S,—SR^(a), ═NR^(a), ═NOR^(a), halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO,—NO₂, ═N₂, —N₃, —S(O)R^(a), —S(O)₂R^(a), —S(O)₂OR^(a), —S(O)NR^(a)R^(a),—S(O)₂NR^(a)R^(a), —OS(O)R^(a), —OS(O)₂R^(a), —OS(O)₂OR^(a),—OS(O)₂NR^(a)R^(a), —C(O)R^(a), —C(O)OR^(a), —C(O)NR^(a)R^(a),—C(NH)NR^(a)R^(a), —OC(O)R^(a), —OC(O)OR^(a), —OC(O)NR^(a)R^(a) and—OC(NH)NR^(a)R^(a).

[0111] In another embodiment, the phenyl xanthene dyes comprise corestructure (II) and, additionally, the substituents therein are definedas follows:

[0112] A is selected from —OH and —NR^(3′)R^(3″);

[0113] B is selected from ═O and ═N^(⊕)R^(6′)R^(6″),;

[0114] R¹, R^(3″), R⁵, R^(6″), R^(6′), R⁷, R⁸, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R^(x), R^(y), R^(a), R^(b), R^(c), R^(d), and R^(e) are as defined withrespect to core structure (I),

[0115] R² is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R² may be taken together with R¹ orR^(2′) to form part of a benzo, naptho or polycyclic aryleno group whichis optionally substituted with one or more of the same or differentR^(a) or suitable R^(b) groups;

[0116] R^(2′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(2′) may be taken together with R² toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, when A is —NR^(3′)R^(3″),R^(2′) may be taken together with R^(3′) to form a 5- or 6-membered ringwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups;

[0117] R^(3′), when present, is selected from hydrogen, (C1-C20) alkylor heteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3′) may be taken together withR^(2′) to form a 5- or 6-membered ring which is optionally substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups;

[0118] R⁴ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, when A is —NR^(3″)R^(3″), R⁴ may betaken together with R^(3″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, R⁴ may be taken togetherwith R^(4′) to form part of a benzo, naptho or polycyclic aryleno groupwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups; and

[0119] R^(4′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(4′) may be taken together with R⁴ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups.

[0120] In another embodiment, the phenyl xanthene dyes comprise corestructure (III) and, additionally, the substituents therein are definedas follows:

[0121] A is selected from —OH and —NR^(3′)R^(3″);

[0122] B is selected from ═O and ═N^(⊕)R^(6′)R^(6″);

[0123] R¹, R^(3″), R^(6″), R⁸, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ R^(x), R^(y),R^(a), R^(b), R^(c), R^(d), and R^(e) are as defined with respect tocore structure (I);

[0124] R², R^(2′), R^(3′), R⁴, and R^(4′) are as defined with respect tocore structure (II);

[0125] R^(5′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) group or, alternatively R⁵ may be taken together with R⁵ to formpart of a benzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups.

[0126] R⁵ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) group or, alternatively, when B is —NR^(3′)R^(3″+), R⁵ may betaken together with R^(6″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, R⁵ may be taken togetherwith R^(5′) to form part of a benzo, naptho or polycyclic aryleno groupwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups;

[0127] R^(6′), when present, is selected from hydrogen, (C1-C20) alkylor heteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(6′) may be taken together withR^(7′) to form a 5- or 6-membered ring optionally substituted with oneor more of the same or different R^(a) or suitable R^(b) groups;

[0128] R^(7′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or R^(b)groups, or, alternatively, when B is —NR^(3′)R^(3″+), R^(7′) may betaken together with R^(6′) to form a 5- or 6-membered ring optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(7′) may be taken together with R⁷ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; and

[0129] R⁷ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or R^(b)groups, or, alternatively, R⁷ may be taken together with R^(7′) or R⁸ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups.

[0130] The list of possible phenyl xanthene dyes is as endless as thevariations that can exist in the upper ring system. However, preferreddyes are fluoresceins, rhodols and rhodamines that have found most usein the industry, as modified to contain the C9 phenyl ring discussedabove. Along this line, the following fluoresceins, rhodols andrhodamines are noted: Rhodamine 101, Rhodamine 110, Rhodamine 6G, TAMRA,ROX, HEX, NAN, FLAN, TET, JOE, and ZOE. Those skilled in the art will beable to name many more commercially important fluoresceins, rhodaminesand rhodols. Any fluorescein, rhodamine or rhodol can be modified at theC9 position to contain the lower phenyl ring.

[0131] 5.4 Lipid Soluble Phenyl Xanthene Dyes

[0132] In one embodiment, the phenyl xanthene dyes not only contain thenew lower phenyl ring but also contain sufficient lipophilic groups tomake the phenyl xanthenes lipid soluble. This is especially beneficialwhen the phenyl xanthene dyes are used, for example, to imbibehydrophobic polymeric particles that are useful in aqueous assays. Suchembodiments are described, for example, in copending patent applicationSer. No. ______, filed concurrently herewith, entitled “FluorescentPolymeric Materials Containing Lipid Soluble Rhodamine Dyes,” thedisclosure of which is incorporated herein by reference.

[0133] Non-limiting examples of such polymeric particles includecrosslinked and uncrosslinked polystyrene particles and styrene-(meth)acrylic acid copolymers. An unlimited variety of particles for use inassays are commercially available, including particles that arefunctionalized and/or paramagnetic and/or conjugated with a biologicalreagents. For example, Bangs Laboratories sells the following products:“plain (hydrophobic) polystyrene microspheres” of various sizes (catalogcodes PS02N, PS03N, PS04N, PS05N, PS06N, PS07N, PS08N, PS00N);“carboxylate-modified microspheres” of various sizes (catalog codesPC02N, PC03N, PC04N, PC05N, PC06N, PC07N, PC08N and PC00N);“amino-modified microspheres” of various sizes (catalog codes PA02N,PA03N, PA04N, PA05N, PA06N, and PA00N); “classical magneticmicrospheres” having carboxlic or amino functionality (catalog codesMC02N, MC03N, MC04N, MC05N, and MC00N); “encapsulated magneticmicrospheres” with carboxylic and amino surface groups (catalog codesME01N, ME02N, ME03N, and ME00N); and “protein-activated” or“protein-coated” microspheres (catalog codes CM01N, CM02N, CM03N, CP01N,CP02N and CP03N). Similarly, Dynal sells Dynabeads® which are uniform,superparamagnetic, monodisperse polymer beads that can either beuncoated or precoated with specific ligands. Dynabeads® are available inthree different sizes, namely, 1 μm (Dynabeads® MyOne™ Streptavidin),2.8 μm (Dynabeads® M-280 and Dynabeads® M-270) and 4.5 μm (Dynabeads®M450 and Dynabeads® M-500).

[0134] In such embodiments, the degree of lipid solubility required forthe phenyl xanthene dye necessarily varies as a function of the polymerutilized, the aqueous solvent or solvent system employed in the assay inwhich the polymeric particle is to be used, and the conditions (e.g.,time, temperature, pressure, pH, etc.) under which the assay is run.Suitable degrees of lipid solubility are easily determined by methodsknown in the art. For example, suitable lipid solubility can bedetermined by a partition test wherein a known quantity of dye inorganic solvent is combined with the aqueous solvent or solvent systemused in the assay. If a partition results and, under the conditions usedin the assay, there is no appreciable crossing by the dye into thesolvent or solvent system, then the dye is sufficiently lipid soluble.Put another way, the lipid soluble phenyl xanthene dye should besufficiently lipid soluble such that it is capable of being imbibed intothe polymer when dissolved in an organic solvent or solvent system and,when the dyed polymer is subjected to the aqueous conditions of theassay, the dye should resist leaching out of the polymer to any degreethat significantly impacts the fluorescent signature of the dye imbibedpolymer or the results of the assay.

[0135] In those embodiments where the phenyl xanthene dyes are lipidsoluble rhodamines, one or both of the exocyclic amine and exocyclicimminium nitrogens are often substituted with a lipophilic groupdesigned to impart to the rhodamine lipophilic characteristics orproperties. Thus, useful dyes include rhodamines that comprise the C9phenyl ring described above and additionally comprise one or twolipophilic substituents at the exocyclic amine nitrogen and/or one ortwo lipophilic substituents at the exocyclic imminium nitrogen. In oneembodiment, both the exocyclic amine nitrogen and the exocyclic imminiumnitrogen are substituted with a lipophilic group. In another embodiment,the exocyclic amine nitrogen and the exocyclic imminium nitrogen areboth substituted with two lipophilic groups. The lipophilic groups,whether attached to the same or different exocyclic nitrogen, may be thesame or different. In one embodiment, the lipophilic groups on theexocyclic nitrogens are the same.

[0136] In those embodiments where the phenyl xanthene dyes are lipidsoluble rhodols, the exocyclic amine nitrogen is often substituted witha lipophilic group designed to impart to the rhodol lipophiliccharacteristics or properties. Thus, useful dyes include rhodols thatcomprise the C9 phenyl ring described above and also comprise one or twolipophilic substituents at the exocyclic amine nitrogen. In oneembodiment, the exocyclic amine nitrogen is substituted with onelipophilic group. In another embodiment, the exocyclic amine nitrogen issubstituted with two lipophilic groups. If there are two lipophilicgroups on the exocyclic amine nitrogen, the lipophilic groups may besame or different. In one embodiment, there are two lipophilic groups onthe exocyclic amine nitrogen that are the same.

[0137] Lipid-soluble phenyl xanthenes may include lipophilicsubstituents at other positions, as well. It is the net effect of thelipophilic substituents that determines whether the phenyl xanthene dyeis lipid soluble. This is especially true for fluoresceins which have noexocyclic amine or imminium nitrogens.

[0138] Lipophilic substituents are groups that impart the resultantphenyl xanthene dye with lipophilic characteristics or properties asdenoted above. The nature of each lipophilic substituent is notcritical, as long as the resultant phenyl xanthene dye is lipid soluble.Non-limiting examples of suitable lipophilic substituents includeunsubstituted (C4-C20) alkyls, (C5-C40) aryls, and (C6-C40) arylalkyls.Depending on the number of methylene and methine units in the lipophilicsubstituent, the lipophilic substituent may also include pendant orinternal polar or hydrophilic groups. For example, a lipophilicsubstituent may include one or more internal heteroatoms, such as one ormore internal O, S, N or NH groups. As another example, a lipophilicsubstituent may include one or more pendant polar or hydrophilicsubstituents, such as one or more pendant halogen, —OH, —SH, —NH₂,—C(O)OH, —C(O)NH₂ or other polar or hydrophilic groups. Thus, lipophilicsubstituents may also include substituted (C4-C20) alkyl, substituted(C5-C40) aryls and substituted (C6-C40) arylalkyls, as well assubstituted and unsubstituted (C4-C20) heteroalkyl, substituted andunsubstituted (C5-C40) heteroaryls and substituted and unsubstituted(C6-C40) arylalkyls. The number of internal or pendant polar orhydrophilic groups that may be included in a lipophilic substituent willdepend upon, among other factors, the number of methylene or methinegroups included in the lipophilic substituent and the number oflipophilic substituents on the phenyl xanthene dye. The nature andnumber of lipophilic groups necessary to make a phenyl xanthene lipidsoluble can vary from molecule to molecule, and will be apparent tothose of skill in the art.

[0139] 5.5 Conjugatable Phenyl Xanthene Dyes

[0140] Oftentimes, it is desirable to attach fluorescent dyes such asthe phenyl xanthene dyes described herein to substances such as solidsupports, particles, and biological and non-biological molecules (e.g.,drugs, amino acids, peptides, polypeptides, proteins, nucleosides,nucleotides, oligonucleotides, polynucleotides, carbohydrates, etc.)Thus, in one embodiment, the various phenyl xanthene dyes describedherein include one or more moieties suitable for such attachment. Suchmoieties are expressed by the formula —S-LG where S is a direct bond ora spacing moiety and LG is a linking group capable of forming a linkagewith the substance to be conjugated.

[0141] The linking group LG may be any moiety capable of forming thelinkage, which may be covalent or non-covalent. For example, the linkinggroup may be one member of a pair of specific binding molecules thatnon-covalently bind one another, such as biotin and avidin/streptavidin.Thus, in one embodiment, the linking group is biotin. Alternatively, thelinking group may be a functional group capable of forming a covalentlinkage with a “complementary” functional group, such as anelectrophilic (or nucleophilic) group which is capable of forming acovalent linkage with a complementary nucleophilic (or electrophilic)group, although other groups may be used depending on the desiredlinking chemistry, as is well known in the art. Non-limiting examples ofsuitable electrophilic linking groups include any one or a combinationof the following: amines/anilines, alcohols/phenols, thiols, hydrazinesand hydroxylamines; Non-limiting examples of suitable electrophiliclinking groups include any one or a combination of the following:activated esters such as pentafluorophenyl ester and NHS-ester,acrylamides, acyl azides, acyl halides, acyl nitriles, aldehydes,ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides,aziridines, boronates, carboxylic acids, carbodiimides, diazoalkenes,epoxides, haloacetamides, halotriazines, imido esters, isocyanates,isothiocyanates, maleimides, phosphoamidites, silyl halides, sulfonateesters and sulfonyl esters.

[0142] The linking group may be attached directly to the phenyl xanthenedye or it may be spaced away from the phenyl xanthene dye by way ofspacing moiety “S.” As will be appreciated by skilled artisans, thenature and composition of the spacing moiety is not critical and maydepend upon the particular application. Thus, the spacing moiety maycomprise virtually any combination of atoms or groups commonly employedto space one molecule from another. As a specific example, the spacingmoiety may be selected from substituted or unsubstituted alkylenes orheteroalkylenes, substituted or unsubstituted arylenes orheteroarylenes, substituted or unsubstituted arylalkylenes orheteroarylalkylenes, or a combination of such groups. In one embodiment,the spacing moiety is an unsubstituted alkylene of the formula—(CH₂)_(n)—, where n is an integer ranging from 1 to 40, typically from1 to 20 and more typically from 1 to 10. Other exemplary spacingmoieties and linking groups are described, for example, in U.S. Pat.Nos. 4,439,356, 4,481,136, 5,188,934, 5,654,442, 5,863,727, 5,847,162,6,229,055, 6,248,884 and 6,372,907.

[0143] The linking group, whether attached directly or spaced away viaspacing moiety “S,” may be attached to any available position of thephenyl xanthene dye. For example, the linking group may be attached toany available position on the upper ring system or the lower ring. Inone embodiment, the linking group —S-LG is attached to the C2, C4, C5,or C7 position of the upper ring system. In another embodiment, thelinking group —S-LG is attached to the C12, C13 or C14 position of thelower ring.

[0144] In one embodiment, a phenyl xanthene suitable for covalentattachment comprises any of the previously-described phenyl xantheneswherein one or more of R¹, R², R^(2′), R⁴, R^(4′), R⁵, R^(5′), R⁷,R^(7′), R⁸, R¹¹, R¹², R¹³, R¹⁴ or R¹⁵ is a substituent of the formula—S-LG, where S is a direct bond or a spacing moiety and LG represents alinking group. In a specific embodiment, one of R⁴, R⁵, R¹², R¹³ or R¹⁴is —S-LG.

[0145] 5.6 Conjugated Phenyl Xanthene Dyes

[0146] The lipid-soluble rhodamine dyes may be linked to anothersubstance. In this embodiment, at least one substituent on the phenylxanthene dye is —S¹-LK-S²—CS. In this case, CS represents the conjugatedsubstance and S¹, LK and S² form what is known in the art as a“linker”—which embraces any functionality known in the art that attachesa dye to another substance.

[0147] S¹ and S² are, independently of one another, a covalent bond or aspacing moiety. The nature of the spacing moieties S¹ and S² may varybroadly. Illustrative spacing moieties include those previouslyspecified for the spacing moiety “S.”

[0148] LK represents a linkage, which may be a bond or another type oflinkage, such as a linkage formed between a nucleophilic (orelectrophilic) group and a complementary electrophilic (or nucleophilic)group. In one embodiment, LK is selected from an ester, an amide, asulfonamide, a hydrazine, an imine, a maleimide, a sulfide, a disulfide,a carbamate and a thiocarbamate linkage.

[0149] The linker will vary depending the identity of the conjugatedsubstance. Illustrative linkers are provided, for example, in U.S. Pat.Nos. 4,439,356, 4,481,136, 5,188,934, 5,654,442, 5,863,727, 5,847,162,6,229,055, 6,248,884 and 6,372,907.

[0150] In one exemplary embodiment, a conjugated phenyl xanthene is anyof the previously-described phenyl xanthenes in which one or more of R¹,R², R^(2′), R⁴, R^(4′), R⁵, R^(5′), R⁷, R^(7′), R⁸, R¹¹, R¹², R¹³, R¹⁴or R¹⁵ is a substituent of the formula —S¹-LK-S²—CS. In a specificembodiment, one of R⁴, R⁵, R¹², R¹³ or R¹⁴ is —S¹-LK-S²—CS.

[0151] 5.7 Energy Transfer Dyes

[0152] In another embodiment, the phenyl xanthene dye is part of anenergy transfer (“ET”) network comprising, for example, from two to fourdyes covalently attached to one another that transfer energy to generatea longer Stoke's shift. In other words, the phenyl xanthene dye may bepart of series of dyes that are covalently attached to one another. Oneexample of an ET network would be a fluorescence resonance energytransfer (“FRET”) dye. In this embodiment, at least one substituent onthe phenyl xanthene dye is selected from —S¹-LK-S²-D, where S¹, S² andLK are as previously defined and D is another dye in the network. In oneembodiment, each dye in the energy transfer network is within 5 to 100 Åof the neighboring dye or dyes in the network to which it is covalentlyattached. In such embodiments, the phenyl xanthene dye can be the donor,acceptor, or an intermediate dye in the network.

[0153] In one embodiment, an energy transfer dye comprises any of thepreviously-described phenyl xanthenes in which one or more of R¹, R²,R^(2′), R⁴, R^(4′), R⁵, R^(5′)R⁷, R^(7′), R⁸, R¹¹, R¹², R¹³, R¹⁴ or R¹⁵is a substituent of the formula —S¹-LK-S²-D. In a specific embodiment,one of R⁴, R⁵, R¹², R¹³ or R¹⁴ is —S¹-LK-S²-D.

[0154] The identity of donor or acceptor dye is not critical, so long asit can donate or accept energy from or to the particular phenyl xantheneto which it is attached. Dyes that can act as donor or acceptors forphenyl xanthenes are well-known, and include, for example, otherfluoresceins, rhodamines, and rhodols, as well as cyanines,phthalocyanine and squaraine dyes. Any of these dyes, or another phenylxanthene as described herein, may be used as the donor dye or acceptordye in an energy transfer dye comprising the phenyl xanthene. Theability to select a suitable dye for a particular phenyl xanthene iswithin the routine skill in the art.

[0155] As will be appreciated by skilled artisans, the varioussubstituents S¹, LK and S of the linker should be selected to positionthe lipid-soluble rhodamine and acceptor or donor dye in close enoughproximity to one another such that the dyes can undergo energy transfer,whether via FRET or another mechanism.

[0156] Suitable linkers are illustrated, for example, by U.S. Pat. Nos.5,800,996 and 5,863,727, issued to Lee et al., U.S. Pat. No. 6,008,279,issued to Benson et al., and U.S. Pat. No. 5,654,419, issued to Mathieset al., all of which are hereby incorporated by reference. Methods ofsynthesizing such energy transfer dyes, as well as suitable points ofattachment for covalently coupling the lipid-soluble rhodamine andacceptor or donor dye D to one another are also described in thesepatents.

[0157] In one exemplary embodiment, an energy transfer dye of theformula D¹-S¹-LK-S²-D², where D¹ represents a phenyl xanthene dye, Drepresents another dye, and S¹, LK, and S² are as defined above, may besynthesized by reacting a phenyl xanthene of the formula D¹-S¹-LG, whereLG represents a linking group, with a donor or acceptor dye of theformula D²—S²-LG′, where LG′ represents a linking group which iscomplementary to linking group LG such that LG and LG′ may react withone another to form linkage LK. As a specific embodiment, LG may be anactivated ester such as an NHS-ester and LG′ may be a primary aminogroup, such that reaction forms an amide linkage LK

[0158] 5.8 Method for Synthesizing Phenyl Xanthene Dyes

[0159] Phenyl xanthene dyes which include a C9-phenyl moiety which doesnot have an ortho carboxylate or sulfonate substituent may be preparedby methods that are illustrated and described in the figures andexamples, respectively, provided herein. In general, an orthosubstituted benzaldehyde, which may or may not be further substituted,is reacted with either a substituted or unsubstituted3-amino-1-hydroxybenzene or a substituted or unsubstituted1,3-dihydroxybenzene or a mixture thereof. This is visually illustratedbelow:

[0160] where A is an amino or hydroxyl group, depending on whether itarises from compound 10 or 20 respectively, where B is an imminium or anoxo group, depending on whether it arises from compound 10 or 20respectively, and where the amine on compound 10 may be furthersubstituted with any desirable substituents for R^(3′), R^(3″), R^(6′)and R^(6″).

[0161] The reaction is carried out in solution, for example suspended in1,2-dichlorbenzene. The reaction is carried out under heat. Generally, atemperature ranging from 160 to 170° C. will suffice. Preferably, acatalyst is employed. For reactions utilizing substituted orunsubstituted 3-amino-1-hydroxybenzene, lithium perchlorate is a goodcatalyst. For reactions employing 1.3-dihydroxyphenol, toluene sulfonicacid is a good catalyst. Under these conditions, the reaction takesabout 60 minutes to complete.

[0162] Suitable aminophenol and benzaldehyde compounds are commerciallyavailable and easily isolated or synthesized by one of skill in the art.For example, benzaldehyde can be made by partial reduction of a benzoicacid, amide, or nitrile. Similarly, 3-amino-1-hydroxybenzene can bemanufactured by the reduction of nitrophenol as described in U.S. Pat.No. 3,079,435. Dihydroxy rescorcinol and the like are commerciallyavailable from Aldrich Chemical Company.

[0163] Extended phenyl xanthenes can be made by utilizing amino-hydroxysubstituted naphthalenes and/or dihydroxy substituted naphthalenes inconjunction with the 3-amino-1-hydroxybenzenes and 1,2-dihydroxybenzenesdiscussed above. This reaction is visually illustrated below:

[0164] where A is an hydroxy or amino group, depending on whether itarises from compound 30 or 40 respectively, where B is an oxo or animminium group, depending on whether it arises from compound 30 or 40respectively, and where the amine on compound 40 may be furthersubstituted with any desirable substituents for R^(3′), R^(3″), R^(6′)and R^(6″).

[0165] 5.9 Improved Properties

[0166] The phenyl xanthene dyes described herein, when excited by alight source, emit an unusually strong spectral signal with lowbackground noise. Lipid soluble embodiments of the xanthene dyes areeasily imbibed into, and retained by, hydrophobic polymeric particles,even in the presence of water based solvents. Finally, the rhodaminedyes are highly photo and chemically stable. In fact, some of therhodamine dyes have photostabilities ten times that of fluorescein and100 times that of cyanine.

[0167] 5.10 Illustrative Uses

[0168] The phenyl xanthene dyes of the instant invention have directapplications in a number of technologies, including use as fluorescentlabels in automated DNA sequencing, oligonucleotide hybridizationmethods, detection of polymerase-chain reaction products, immunoassays,and the like. For many applications, multiple dyes are employed, incombination, to permit multiplex fluorescent detection.

[0169] The phenyl xanthene dyes can be used to sequence nucleic acidsfor example using the Sanger method. The specifics of sequencing nucleicacids by the Sanger method are well-known in the art and are notrepeated here. For such sequencing applications, the phenyl xanthenedyes described herein may be attached to the primer or to a terminatingnucleotide, such as a 2,3′-dideoxynucleotide triphosphate. Examples ofvarious labeled primers, labeled terminating nucleotides and methods ofusing such labeled primers and terminating nucleotides in sequencing andother applications are described in U.S. Pat. Nos. 5,188,934, 5,366,860,5,654,442, 5,800,996, 5,840,999, 5,847,162, 5,863,727, 5,936,087,6,008,379, 6,248,884 and 6,372,907, the disclosures of which areincorporated herein by reference. The xanthene dyes described herein maybe attached to similar primers and terminating nucleotides and used inan analogous manner.

[0170] Alternatively, the phenyl xanthene dyes can be imbibed intoparticles used in the passive or covalent coupling of analytes. In aparticularly preferred aspect of the invention, a mixture of lipidsoluble phenyl xanthenes and, optionally additional dyes, are internallyincorporated, simultaneously or sequentially, into polymericmicroparticles to give the microparticles a unique spectral signature or“bar code.” A number of particle populations are created, eachcharacterized by a different spectral bar code. The particles can thenbe activated or otherwise modified so that they have a specificreactivity with one or more analytes in a clinical or test sample. Thus,the spectral bar code in each particle population corresponds to adifferent known reactivity. The particle populations can then be blendedin a specified ratio to form a multicolored particle mixture which isthen contacted with the analyte. Imbibed bead mixtures may containhundreds to thousands of fluorescent dye molecules which greatlyincreases the sensitivity of assays employing bead labels in comparisonto single dye assays.

[0171] To achieve truly multiplexed analysis of a plurality of analytesin a single sample, some sort of additional marker is necessary to showthat a positive event has occurred on a particle. This additional markercan be many things, for example, it can be a molecule, such as biotin,which is detectable by its interaction with another compound, in thisexample streptavidin. Alternatively, the additional marker can be secondfluorescent signal, e.g., a green fluorescent label. The marker is oftenprovided by a labeling reagent which is also capable of binding to theanalyte of interest.

[0172] 5.11 Inherent Limitations in Structures

[0173] Those skilled in the art will appreciate that many of the phenylxanthene dye compounds described in the various structures herein mayexhibit the phenomena of tautomerism, conformational isomerism,geometric isomerism and/or stereo isomerism. As the structures presentedin the specification and claims can represent only one tautomeric,conformational isomeric, enantiomeric or geometric isomeric form, itshould be understood that the invention encompasses any tautomeric,conformational isomeric, enantiomeric and/or geometric isomeric forms ofthe compounds that have one or more of the utilities described herein.As a specific example, reference is made throughout the specification tothe C3 amino and C6 imminium substituents in rhodamines and rhodols. Asthis nomenclature corresponds to the illustrated structures, whichrepresent only one of several possible tautomeric forms (or resonancestructures) of the compounds, it will be understood that thesereferences are for convenience only and that any such references are notintended to limit the scope of the compounds described herein.

[0174] Furthermore, those of skill in the art will recognize that thephenyl xanthene dyes of the invention may exist in many differentprotonation states, depending on, among other things, the pH of theirenvironment The structures provided herein depict the compounds in onlyone of several possible protonation states. Accordingly, it will beunderstood that these structures are illustrative only, and that theinvention is not limited to any particular protonation state—any and allprotonated forms of the dyes are intended to fall within the scope ofthe invention.

[0175] As the phenyl xanthene dye compounds used in the invention maybear positive and negative charges, depending upon their physical state,they often have counterions associated therewith. The identity oridentities of any associated counterions is typically dictated by thesynthesis and/or isolation methods by which the compounds are obtained.Typical counterions include, but are not limited to, halides, acetate,trifluoroacetate, any salt of a strong acid, and mixtures thereof. Itwill be understood that the identity or identities of any associatedcounterions are not a critical feature of the invention and that theinvention encompasses the use of dyes in association with any type ofcounter ion. Moreover, as the compounds can exists in a variety ofdifferent forms, the invention is intended to encompass not only formsof the dyes that are in association with counterions (e.g., dry salts),but also forms that are not in association with counterions (e.g.,aqueous or organic solutions).

[0176] 5.12 Incorporation by Reference

[0177] All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference. No admission is made that any reference cited in thisspecification is prior art.

6. EXAMPLES

[0178] 6.1 Overview of Synthesis for Fluorescein, Rhodol and RhodamineDyes

[0179] Exemplary phenyl xanthenes were synthesized from reactions ofsubstituted and unsubstituted 1-hydroxy-3-aminobenzene derivativesand/or substituted and unsubstituted 1,3 dihydroxybenzene derivativeswith phenyl aldehyde derivatives. Dye structures were verified by massspectrometry.

6.2 Example 1

[0180] As illustrated in FIG. 1A, phenyl xanthenes containingsymmetrically substituted upper ring systems were made by reacting oneor more 3-amino-1-hydroxy-benzenes 120 (where y^(A,B) is an amine)and/or one or more 1,3-dihydroxy-benzene 120 (where Y^(A,B) is ahydroxyl) with a phenyl aldehyde of general structure 130. Reactantswere suspended in a high boiling solvent, such as dichlorobenzene in thepresence of a catalyst. Acid catalysts, such as toluene sulfonic acid,are used when y^(A,B) is a hydroxyl. Catalysts such as lithiumperchlorate are used when y^(A,B) is an amine. Reactants were heated andstirred for 1 to 5 hours at a temperature from 130° to 155° C.

[0181] By this method, dyes corresponding to structure 140 were made:

[0182] where R¹═R⁸, R²═R⁷ and R⁴═R⁵. Symmetrically substitutedrhodamines were produced by reacting a 3-amino-1-hydroxy-benzene with abenzaldehyde in the presence of lithium perchlorate. Symmetricallysubstituted fluoresceins were produced by reacting a1,3-dihydroxy-benzene with a benzaldehyde in the presence of toluenesulfonic acid.

[0183] Phenyl xanthenes with non-symmetrically substituted upper ringsystems can also be produced by employing multiple3-amino-1-hydroxy-benzenes and/or multiple 1,3-dihydroxy-benzenes thatcontain different substituents.

[0184] Dyes that were made by this procedure include the following:

6.3 Example 2

[0185] As illustrated in FIG. 2A, phenyl xanthenes with symmetricallysubstituted and symmetrically extended upper ring systems were made byreacting an amino-hydroxy-substituted napthalene 220 (where y^(A,B) isan amine) and/or a dihydroxy substituted naphthalene 220 (where y^(A,B)is a hydroxyl) with a phenyl aldehyde of general structure 130.Reactants were suspended in a high boiling solvent, such asdichlorobenzene, in the presence of a catalyst. Acid catalysts, such astoluene sulfonic acid, are used when y^(A,B) is a hydroxyl. Catalystssuch as lithium perchlorate are used when y^(A,B) is an amine. Reactantswere heated and stirred for 1 to 5 hours at a temperature from 130° to155° C.

[0186] By this method, symmetrically extended phenyl xanthene dyescorresponding to structure 240 were made:

[0187] where R¹═R⁸, R²═R⁷, R^(2′)═R⁷, R⁴═R⁵, and R^(4′)═R^(5′).Symmetrially extended and symmetrically substituted rhodamines wereproduced by reacting an amino-hydroxy-substituted napthalene with abenzaldehyde in the presence of an toluene suflonic acid. Symmetriallyextended and symmetrically substituted fluoresceins were produced byreacting a dihydroxy substituted naphthalene with a benzaldehyde in thepresence of lithium perchlorate.

[0188] As illustrated in FIG. 2B, phenyl xanthenes withnon-symmetrically extended upper ring systems can be synthesized by thesame procedure if an additional reactant, namely a3-amino-1-hydroxy-benzene (where y^(A,B) is an amine) and/or a1,3-dihydroxy-benzene 120 (where y^(A,B) is a hydroxyl), is added to thereaction mixture.

[0189] Furthermore, phenyl xanthene dyes with non-symmetricallysubstituted upper ring systems can be synthesized by using a mixture ofreactants with varying substituents as previously described.

[0190] Dyes that were made by this procedure include the following:

6.4 Example 3

[0191] Flourescein dyes of structure 340 were synthesized in reactionsof dihydroxy benzene derivatives 320 and benzoate ester derivatives 100as outlined in FIG. 3. Derivatives of 320 were synthesized byestablished procedures (see U.S. Pat. Nos. 5,188,934 and 6,008,379). Asa general dye synthesis procedure 120 and benzoate ester 130 weresuspended in neat methane sulfonic acid and the reaction heated at 130°C. for 3 hours. The reaction mixture was precipitated by pouring intoice, the solid precipitate collected by filtration, the crude dye 340suspended in CH₂Cl₂/MeOH/AcOH (95:5:0.5), and loaded into a silica gelcolumn. The column was first eluted with CH₂Cl₂/MeOH/AcOH (95:5:0.5) andthen CH₂Cl₂/MeOH/AcOH (80:20:0.5). The fractions containing dye 340 werecombined and concentrated to a solid.

[0192] Alternatively, fluorescein dyes 340 were synthesized fromreactions of benzaldehyde derivatives 130 in dichlorobenezene with 10equivalents of p-toluene sulfonic acid and the reaction mixture washeated with stirring at 130° C. for 3 hours.

6.5 Example 4

[0193] Extended fluorescein dyes of structure 440 were synthesized inreactions of 1,6-dihydroxy napthalene derivatives 320 and benzaldehydederivatives as outlined in FIG. 4. Following the alternative general dyesynthesis conditions described for 340, 1,6-dihydroxynathalene 420 andbenzaldehyde derivatives 130 were suspended in dichlorobenezene with 10equivalents of p-toluene sulfonic acid and the reaction mixture washeated with stirring at 130° C. for 3 hours.

[0194] Following this general procedure, dye 19 below was produced fromreactions of 1,6-dihydroxynapthalene 420 with compounds 130 whereR11-R15=fluorine.

6.6 Example 5

[0195] Rhodol dyes of general structure 540 were synthesized fromreactions of equal equivalents aminohydroxy benzene derivatives 120 oraminonapthol derivatives 220, dihydroxy benzene derivatives 320 ordihydroxynapthalene derivatives 420, and phenyl aldehydes 130, followingthe general procedure described for synthesis of 140 outlined in FIG. 5.

We claim:
 1. A fluorescent phenyl xanthene dye comprising a fluorescein,rhodol or rhodamine that comprises a C9 phenyl ring comprising thefollowing structure:

where R¹¹ and R¹⁵ are independently selected from alkyl, heteroalkyl,alkoxy, halo, haloalkyl, amino, alkylthio, cyano, isocyano, cyanato,mercaptocyanato, nitro, and sulfinyl and R¹², R¹³, and R¹⁴ are,independently, selected from hydrogen and any substituent having up to40 atoms.
 2. The phenyl xanthene dye of claim 1 wherein R¹², R¹³, andR¹⁴ are independently selected from hydrogen, alkyl, heteroalkyl, aryl,heteroaryl, alkoxy, halo, haloalkyl, amino, alkylthio, cyano, isocyano,cyanato, mercaptocyanato, nitro, sulfinyl, sulfonyl, sulfonamide,carboxyl, and carboxyamide.
 3. The phenyl xanthene dye of claim 1wherein at least one of R¹¹ and R¹⁵ is selected from an alkoxy, a halo,a haloalkyl and/or a nitrile.
 4. The phenyl xanthene dye of claim 1wherein R¹¹ and R¹⁵ are selected, independently, from alkoxy, haloand/or haloalkyl groups.
 5. The phenyl xanthene dye of claim 1 whereinthe phenyl ring is only substituted with hydrogen, alkoxy, halo andhaloalkyl groups.
 6. The phenyl xanthene dye of claim 1 wherein R¹¹ andR¹⁵ are identical.
 7. The phenyl xanthene dye of claim 1 wherein thephenyl ring is symmetrical relative to an imaginary axis running fromthe phenyl ring's point of attachment through a point para toattachment.
 8. The phenyl xanthene dye of claim 1 wherein R¹¹ and R¹⁵are identical and R¹², R¹³, and R¹⁴ are, identically, selected fromhydrogen or a substituent that is different from R¹¹ and R¹⁵.
 9. Thephenyl xanthene dye of claim 1 wherein R¹¹, R¹³ and R¹⁵ are identicalfirst substituents and R¹² and R¹⁴ are, identically, selected fromhydrogen or a substituent different from R¹¹, R¹³ and R¹⁵.
 10. Thephenyl xanthene dye of claim 1 wherein R¹¹, R¹², R¹⁴ and R¹⁵ are and R¹³is selected from hydrogen or a substituent different from R¹¹, R¹², R¹⁴and R¹⁵.
 11. The phenyl xanthene dye of claim 1 wherein R¹¹, R¹², R¹³,R¹⁴ and R¹⁵ are identical.
 12. The phenyl xanthene dye of claim 1wherein said dye is a fluorescein.
 13. The phenyl xanthene dye of claim1 wherein said dye is a rhodol.
 14. The phenyl xanthene dye of claim 1wherein said dye is a rhodamine.
 15. The phenyl xanthene dye of claim 1wherein the dye is lipid soluble due to the presence of one or morelipophilic groups.
 16. The phenyl xanthene dye of claim 15 wherein thelipophilic groups are selected from (C4-C20) alkyls, (C5-C40) aryls,and/or (C6-C40) arylalkyls.
 17. The phenyl xanthene dye of claim 15wherein said dye is a rhodamine and at least one of the exocyclic amineand the exocyclic imminium nitrogens are substituted with one or morelipophilic groups.
 18. The phenyl xanthene dye of claim 15 wherein saiddye is a rhodol and the exocyclic amine nitrogen is substituted with oneor more lipophilic groups.
 19. The phenyl xanthene dye of claim 1wherein said dye contains one or more linkers that contain a linkinggroup capable of forming a covalent or non-covalent linkage to a secondsubstance.
 20. The phenyl xanthene dye of claim 19 wherein said secondsubstance is selected from a biomolecule, a glass substrate, a metalsubstrate, and a polymer substrate.
 21. The phenyl xanthene dye of claim19 wherein said second substance is another dye and the phenyl xanthenedye is part of an energy transfer network.
 22. The phenyl xanthene dyeof claim 1 wherein said dye is linked to at least one second substance.23. The phenyl xanthene dye of claim 22 wherein said second substance isselected from a biomolecule, a glass substrate, a metal substrate, and apolymer substrate.
 24. The phenyl xanthene dye of claim 22 wherein saidsecond substance is another dye and the phenyl xanthene dye is part ofan energy transfer network.
 25. A lipid soluble fluorescent phenylxanthene dye comprising a fluorescein, rhodol or rhodamine thatcomprises a C9 phenyl ring comprising the following structure:

where R¹¹ and/or R¹⁵ are selected from alkyl, heteroalkyl, alkoxy, halo,haloalkyl, amino, alkylthio, cyano, isocyano, cyanato, mercaptocyanato,nitro, and sulfinyl, and R¹², R¹³, and R¹⁴ are, independently, selectedfrom hydrogen and any substituent having up to 40 atoms.
 26. The lipidsoluble phenyl xanthene dye of claim 25 where R¹¹ and R¹⁵ are selectedfrom alkyl, heteroalkyl, alkoxy, halo, haloalkyl, amino, alkylthio,cyano, isocyano, cyanato, mercaptocyanato, nitro, and sulfinyl.
 27. Thelipid soluble phenyl xanthene dye of claim 25 wherein R¹², R¹³, and R¹⁴are, independently, selected from hydrogen, alkyl, heteroalkyl, aryl,heteroaryl, alkoxy, halo, haloalkyl, amino, alkylthio, cyano, isocyano,cyanato, mercaptocyanato, nitro, sulfinyl, sulfonyl, sulfonamide,carboxyl, and carboxyamide.
 28. The lipid soluble phenyl xanthene dye ofclaim 25 wherein at least one of R¹¹ and R¹⁵ is selected from an alkoxy,halo, haloalkyl and nitrile.
 29. The lipid soluble phenyl xanthene dyeof claim 25 wherein both of R¹¹ and R¹⁵ are selected, independently,from alkoxy, halo and haloalkyl groups.
 30. The lipid soluble phenylxanthene dye of claim 25 wherein the phenyl ring is only substitutedwith hydrogen, alkoxy, halo, haloalkyl and halo groups.
 31. The lipidsoluble phenyl xanthene dye of claim 25 wherein R¹¹ and R¹⁵ areidentical.
 32. The lipid soluble phenyl xanthene dye of claim 25 whereinthe phenyl ring is symmetrical relative to an imaginary axis runningfrom the phenyl ring's point of attachment through a point para to theattachment.
 33. The lipid soluble phenyl xanthene dye of claim 25wherein R¹¹ and R¹⁵ are identical and R¹², R¹³, and R¹⁴ are,identically, selected from hydrogen and a substituent that is differentfrom R¹¹ and R¹⁵.
 34. The lipid soluble phenyl xanthene dye of claim 25wherein R¹¹, R¹³ and R¹⁵ are identical and R¹² and R¹⁴ are, identically,selected from hydrogen and a substituent that is different from R¹¹, R¹³and R¹⁵.
 35. The lipid soluble phenyl xanthene dye of claim 25 whereinR¹¹ , R¹², R¹⁴ and R¹⁵ are identical and R¹³ is selected from hydrogenor a substituent different from R¹¹, R¹², R¹⁴ and R¹⁵.
 36. The lipidsoluble phenyl xanthene dye of claim 25 wherein R¹¹, R¹², R¹³, R¹⁴ andR¹⁵ are identical.
 37. The lipid soluble phenyl xanthene dye of claim 25wherein said dye is a fluorescein.
 38. The lipid soluble phenyl xanthenedye of claim 25 wherein said dye is a rhodol.
 39. The lipid solublephenyl xanthene dye of claim 25 wherein said dye is a rhodamine.
 40. Thelipid soluble phenyl xanthene dye of claim 25 wherein the lipophilicgroups are selected from (C4-C20) alkyls, (C5-C40) aryls, and/or(C6-C40) arylalkyls.
 41. The lipid soluble phenyl xanthene dye of claim40 wherein said dye is a rhodamine in which one or both of the exocyclicamine and the exocyclic imminium nitrogens are substituted with one ortwo lipophilic groups.
 42. The lipid soluble phenyl xanthene dye ofclaim 40 wherein said dye is a rhodol in which the exocyclic aminenitrogen is substituted with one or two lipophilic groups.
 43. The lipidsoluble phenyl xanthene dye of claim 40 wherein said dye contains one ormore linkers that contain a linking group capable of forming a covalentor a non-covalent linkage to a second substance.
 44. The lipid solublephenyl xanthene dye of claim 43 wherein said second substance isselected from a biomolecule, a glass substrate, a metal substrate and apolymer substrate.
 45. The lipid soluble phenyl xanthene dye of claim 43wherein said second substance is another dye and the phenyl xanthene dyeis part of an energy transfer network.
 46. The lipid soluble phenylxanthene dye of claim 25 wherein said dye is linked to at least onesecond substance.
 47. The lipid soluble phenyl xanthene dye of claim 46wherein said second substance is selected from a biomolecule, a glasssubstrate, a metal substrate, and a polymer substrate.
 48. The lipidsoluble phenyl xanthene dye of claim 46 wherein said second substance isanother dye and the phenyl xanthene dye is part of an energy transfernetwork.
 47. A fluorescent phenyl xanthene dye comprising one of thefollowing core structures:

where A is —OH or NR³⁴⁰ R^(3″), where B is a ═O or ═N^(⊕)R^(6′)R^(6″);where at least one of R¹¹ and R¹⁵ is selected from alkyl, heteroalkyl,alkoxy, halo, haloalkyl, amino, alkylthio, cyano, isocyano, cyanato,mercaptocyanato, nitro, and sulfinyl, wherein one or more of R², R^(2′),R^(3′), R^(3″), R⁴, R^(4′), R⁵, R^(5′), R^(6′), R^(6″), R⁷, R^(7′), R¹²,R¹³, and R¹⁴ are optionally —S-LG or —S¹-LK-S²—CS, where S is a directbond or a spacing moiety, LG is a linking group, S¹ and S² are,independently, a direct bond or a spacing moiety, LK is a linkage formedfrom a reacted linking group, and CS is a conjugated substance; and theremainder of R¹, R², R^(2′), R^(3′), R^(3″), R⁴, R^(4′), R⁵, R^(5′),R^(6′), R^(6″), R⁷, R^(7′), R⁸, R¹², R¹³, and R¹⁴ are, independently,selected from hydrogen and a substituent having less than 40 atoms. 48.The phenyl xanthene dye of claim 47 wherein the dye comprises corestructure (I) and the substituents therein are further defined asfollows: R¹ is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R¹ may be taken together with R² formpart of a benzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R² is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R² may be taken together with R¹, toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, when A is —NR^(3′)R^(3″),R² may be taken together with R^(3′) to form a 5- or 6-membered ringwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups; R^(3′), when present, isselected from hydrogen, (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroarylalkyl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups, or, alternatively,R^(3′) may be taken together with R² to form a 5- or 6-membered ringwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups; R^(3″), when present, isselected from (C1-C20) alkyl or heteroalkyl optionally substituted withone or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R^(3″) maybe taken together with R⁴ to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R⁴ is selected from hydrogen, R^(x), (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, when B is —NR^(3′)R^(3″), R⁴ may betaken together with R^(3″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R⁵ is selected from hydrogen, R^(x), (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) group or, alternatively, when B is —NR^(6′)R^(6″+), R⁵ may betaken together with R^(6″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R^(6″), when present, is selected from(C1-C20) alkyl or heteroalkyl optionally substituted with one or more ofthe same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups or, alternatively R^(6″) may be taken togetherwith R⁵ to form a 5- or 6-membered ring which is optionally substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups; R^(6′), when present, is selected from hydrogen, (C1-C20) alkylor heteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(6′) may be taken together with R⁷ toform a 5- or 6-membered ring optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups; R⁷ is selectedfrom hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroaryl alkyl optionally substituted with one or more ofthe same or different R^(a) or R^(b) groups, or, alternatively, R⁷ maybe taken together with R⁸ to form part of a benzo, naptho or polycyclicaryleno group which is optionally substituted with one or more of thesame or different R^(a) or suitable R^(b) groups, or, alternatively,when B is —NR^(6′)R^(6″+), R⁷ may be taken together with R^(6′) to forma 5- or 6-membered ring optionally substituted with one or more of thesame or different R^(a) or suitable R^(b) groups; R⁸ is selected fromhydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionally substitutedwith one or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R⁸ togetherwith R⁷ may form part of a benzo, naptho or polycyclic aryleno groupwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups; R¹¹ and R¹⁵ are each,independently of one another, selected from halo, (C1-C20) alkyl,haloalkyl, —OR^(y), —SR^(y), —SOR^(y), —SO₂R^(y), and nitrile; R¹², R¹³and R¹⁴ are each, independently of one another, selected from hydrogen,R^(x), (C1-C20) alkyl or heteroalkyl optionally substituted with one ormore of the same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R^(x) is selected from —NR^(c)R^(c), —OR^(d),—SR^(d), halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃,—S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and—OC(NH)NR^(c)R^(c); R^(y) is selected from (C1-C20) alkyls orheteroalkyls optionally substituted with lipophilic substituents,(C5-C20) aryls or heteroaryls optionally substituted with lipophilicsubstituents and (C2-C26) arylalkyl or heteroarylalkyls optionallysubstituted with lipophilic substituents; R^(a) is selected fromhydrogen, (C1-C8) alkyl or heteroalkyl, (C5-C20) aryl or heteroaryl and(C6-C28) arylalkyl or heteroarylalkyl; R^(b) is selected from—NR^(c)R^(c), ═O, —OR^(d), ═S, —SR^(d), ═NR^(d), ═NOR^(d), halo,haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and —OC(NH)NR^(c)R^(c);each R^(c) is independently hydrogen or R^(d), or, alternatively, eachR^(c) is taken together with the nitrogen atom to which it is bonded toform a 5 to 8-membered saturated or unsaturated ring which mayoptionally include one or more of the same or different additionalheteroatoms and which may optionally be substituted with one or more ofthe same or different R^(a) or R^(d) groups; each R^(d) is independentlyR^(a) or R^(a) substituted with one or more of the same or differentR^(a) or R^(e) groups; and each R^(e) is selected from —NR^(a)R^(a), ═O,—OR^(a), ═S, —SR^(a), ═NR^(a), ═NOR^(a), halo, haloalkyl, —CN, —NC,—OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(a), —S(O)₂R^(a), —S(O)₂OR^(a),—S(O)NR^(a)R^(a), —S(O)₂NR^(a)R^(a), —OS(O)R^(a), —OS(O)₂R^(a),—OS(O)₂OR^(a), —OS(O)₂NR^(a)R^(a), —C(O)R^(a), —C(O)OR^(a),—C(O)NR^(a)R^(a), —C(NH)NR^(a)R^(a), —OC(O)R^(a), OC(O)OR^(a),—OC(O)NR^(a)R^(a) and —OC(NH)NR^(a)R^(a) or, alternatively, one or moreof R², R^(3′), R^(3″), R⁴, R⁵, R^(6′), R^(6″), R⁷, R¹², R¹³ and R¹⁴ areeach, independently of one another, selected from the group consistingof —S-LG and —S¹-LK-S²—CS, wherein each S, which may be the same ordifferent, is a direct bond or linker; each LG, which may be the same ordifferent, is a linking group; each S¹ and S², independently, is adirect bond or linker; each LK is a linkage formed from a reactedlinking group, and each CS, which may be the same or different is aconjugated substance.
 49. The phenyl xanthene dye of claim 47 whereinthe dye comprises core structure (II) and, additionally, thesubstituents therein are defined as follows: R¹ is selected fromhydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionally substitutedwith one or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R¹ may betaken together with R² form part of a benzo, naptho or polycyclicaryleno group which is optionally substituted with one or more of thesame or different R^(a) or suitable R^(b) groups; R² is selected fromhydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionally substitutedwith one or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R² may betaken together with R¹ or R^(2′) to form part of a benzo, naptho orpolycyclic aryleno group which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups; R^(2′) isselected from hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroaryl alkyl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups, or, alternatively,R^(2′) may be taken together with R² to form part of a benzo, naptho orpolycyclic aryleno group which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups, or,alternatively, when A is —NR^(3′)R^(3″), R^(2′) may be taken togetherwith R^(3′) to form a 5- or 6-membered ring which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R^(3′), when present, is selected from hydrogen, (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3′) may be taken together withR^(2′) to form a 5- or 6-membered ring which is optionally substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups; R^(3″), when present, is selected from (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3″) may be taken together with R⁴ toform a 5- or 6-membered ring which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups; R⁴ isselected from hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroarylalkyl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups, or, alternatively,when A is —N R^(3′)R^(3″), R⁴ may be taken together with R^(3″) to forma 5- or 6-membered ring which is optionally substituted with one or moreof the same or different R^(a) or suitable R^(b) groups, or,alternatively, R⁴ may be taken together with R^(4′) to form part of abenzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R^(4′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(4′) may be taken together with R⁴ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, R⁵ is selected from hydrogen, R^(x), (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) group or, alternatively, when B is —NR^(6′)R^(6″+), R⁵ may betaken together with R^(6″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R^(6″), when present, is selected from(C1-C20) alkyl or heteroalkyl optionally substituted with one or more ofthe same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups or, alternatively R^(6″) may be taken togetherwith R⁵ to form a 5- or 6-membered ring which is optionally substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups; R^(6′), when present, is selected from hydrogen, (C1-C20) alkylor heteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(6′) may be taken together with R⁷ toform a 5- or 6-membered ring optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups; R⁷ is selectedfrom hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroaryl alkyl optionally substituted with one or more ofthe same or different R^(a) or R^(b) groups, or, alternatively, R⁷ maybe taken together with R⁸ to form part of a benzo, naptho or polycyclicaryleno group which is optionally substituted with one or more of thesame or different R^(a) or suitable R^(b) groups, or, alternatively,when B is —NR^(6′)R^(6″+), R⁷ may be taken together with R^(6′) to forma 5- or 6-membered ring optionally substituted with one or more of thesame or different R^(a) or suitable R^(b) groups; R⁸ is selected fromhydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionally substitutedwith one or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R⁸ togetherwith R⁷ may form part of a benzo, naptho or polycyclic aryleno groupwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups; R¹¹ and R¹⁵ are each,independently of one another, selected from halo, (C1-C20) alkyl,haloalkyl, —OR^(y), —SR^(y), —SOR^(y), —SO₂R^(y), and nitrile; R¹², R¹³and R¹⁴ are each, independently of one another, selected from hydrogen,R^(x), (C1-C20) alkyl or heteroalkyl optionally substituted with one ormore of the same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R^(x) is selected from —NR^(c)R^(c), —OR^(d),—SR^(d), halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂,—N₃,—S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and—OC(NH)NR^(c)R^(c); R^(y) is selected from (C1-C20) alkyls orheteroalkyls optionally substituted with lipophilic substituents,(C5-C20) aryls or heteroaryls optionally substituted with lipophilicsubstituents and (C2-C26) arylalkyl or heteroarylalkyls optionallysubstituted with lipophilic substituents; R^(a) is selected fromhydrogen, (C1-C8) alkyl or heteroalkyl, (C5-C20) aryl or heteroaryl and(C6-C28) arylalkyl or heteroarylalkyl; R^(b) is selected from—NR^(c)R^(c), ═O, —OR^(d), ═S, —SR^(d), ═NR^(d), ═NOR^(d), halo,haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and —OC(NH)NR^(c)R^(c);each R^(c) is independently hydrogen or R^(d), or, alternatively, eachR^(c) is taken together with the nitrogen atom to which it is bonded toform a 5 to 8-membered saturated or unsaturated ring which mayoptionally include one or more of the same or different additionalheteroatoms and which may optionally be substituted with one or more ofthe same or different R^(a) or R^(d) groups; each R^(d) is independentlyR^(a) or R^(a) substituted with one or more of the same or differentR^(a) or R^(e) groups; and each R^(e) is selected from —NR^(a)R^(a), ═O,—OR^(a), ═S, —SR^(a), ═NR^(a), ═NOR^(a), halo, haloalkyl, —CN, —NC,—OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(a), —S(O)₂R^(a), —S(O)₂OR^(a),—S(O)NR^(a)R^(a), —S(O)₂NR^(a)R^(a), —OS(O)R^(a), —OS(O)₂R^(a),—OS(O)₂OR^(a), —OS(O)₂NR^(a)R^(a), —C(O)R^(a), C(O)OR^(a),C(O)NR^(a)R^(a), —C(NH)NR^(a)R^(a), —OC(O)R^(a), —OC(O)OR^(a),—OC(O)NR^(a)R^(a) and —OC(NH)NR^(a)R^(a), or, alternatively, one or moreof R², R^(2′), R^(3′), R^(3″), R⁴, R⁵ , R^(6′), R^(6″), R⁷, R¹², R¹³ andR¹⁴ are each, independently of one another, selected from the groupconsisting of —S-LG and —S¹-LK-S²—CS, wherein each S, which may be thesame or different, is a direct bond or linker, each LG, which may be thesame or different, is a linking group; each S¹ and S², independently, isa direct bond or linker, each LK is a linkage formed from a reactedlinking group, and each CS, which may be the same or different is aconjugated substance.
 50. The phenyl xanthene dye of claim 47 whereinthe dye comprises core structure (III) and, additionally, thesubstituents therein are defined as follows: R¹ is selected fromhydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionally substitutedwith one or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(b) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R¹ may betaken together with R² form part of a benzo, naptho or polycyclicaryleno group which is optionally substituted with one or more of thesame or different R^(a) or suitable R^(b) groups; R² is selected fromhydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionally substitutedwith one or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroaryl alkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R² may betaken together with R¹ or R^(2′) to form part of a benzo, naptho orpolycyclic aryleno group which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups; R^(2′) isselected from hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroaryl alkyl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups, or, alternatively,R^(2′) may be taken together with R² to form part of a benzo, naptho orpolycyclic aryleno group which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups, or,alternatively, when A is —NR^(3′)R^(3″), R^(2′) may be taken togetherwith R^(3′) to form a 5- or 6-membered ring which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R^(3′), when present, is selected from hydrogen, (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3′) may be taken together withR^(2′) to form a 5- or 6-membered ring which is optionally substitutedwith one or more of the same or different R^(a) or suitable R^(b)groups; R^(3″), when present, is selected from (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(3″) may be taken together with R⁴ toform a 5- or 6-membered ring which is optionally substituted with one ormore of the same or different R^(a) or suitable R^(b) groups; R⁴ isselected from hydrogen, R^(x), (C1-C20) alkyl or heteroalkyl optionallysubstituted with one or more of the same or different R^(b) groups,(C5-C20) aryl or heteroaryl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups and (C6-C40)arylalkyl or heteroarylalkyl optionally substituted with one or more ofthe same or different R^(a) or suitable R^(b) groups, or, alternatively,when A is —NR^(3′)R^(3″), R⁴ may be taken together with R^(3″) to form a5- or 6-membered ring which is optionally substituted with one or moreof the same or different R^(a) or suitable R^(b) groups, or,alternatively, R⁴ may be taken together with R^(4′) to form part of abenzo, naptho or polycyclic aryleno group which is optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R^(4′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(4′) may be taken together with R⁴ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, R^(5′) is selected from hydrogen, R^(x),(C1-C20) alkyl or heteroalkyl optionally substituted with one or more ofthe same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroarylalkyloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) group or, alternatively R⁵ may be taken together withR⁵ to form part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R⁵ is selected from hydrogen, R^(x), (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) group or, alternatively, when B is —NR^(3′)R^(3″+), R⁵ may betaken together with R^(6″) to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, R⁵ may be taken togetherwith R^(5′) to form part of a benzo, naptho or polycyclic aryleno groupwhich is optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups; R^(6″), when present, isselected from (C1-C20) alkyl or heteroalkyl optionally substituted withone or more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroarylalkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups or, alternatively R^(6″) may betaken together with R⁵ to form a 5- or 6-membered ring which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R^(6′), when present, is selected fromhydrogen, (C1-C20) alkyl or heteroalkyl optionally substituted with oneor more of the same or different R^(b) groups, (C5-C20) aryl orheteroaryl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups and (C6-C40) arylalkyl orheteroarylalkyl optionally substituted with one or more of the same ordifferent R^(a) or suitable R^(b) groups, or, alternatively, R^(6′) maybe taken together with R^(7′) to form a 5- or 6-membered ring optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R^(7′) is selected from hydrogen, R^(x), (C1-C20) alkyl orheteroalkyl optionally substituted with one or more of the same ordifferent R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyl optionallysubstituted with one or more of the same or different R^(a) or R^(b)groups, or, alternatively, when B is —NR^(3′)R^(3″+), R^(7′) may betaken together with R^(6′) to form a 5- or 6-membered ring optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups, or, alternatively, R^(7′) may be taken together with R⁷ toform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; and R⁷ is selected from hydrogen, R^(x),(C1-C20) alkyl or heteroalkyl optionally substituted with one or more ofthe same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyloptionally substituted with one or more of the same or different R^(a)or R^(b) groups, or, alternatively, R⁷ may be taken together with R^(7′)or R⁸ to form part of a benzo, naptho or polycyclic aryleno group whichis optionally substituted with one or more of the same or differentR^(a) or suitable R^(b) groups. R⁸ is selected from hydrogen, R^(x),(C1-C20) alkyl or heteroalkyl optionally substituted with one or more ofthe same or different R^(b) groups, (C5-C20) aryl or heteroaryloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups and (C6-C40) arylalkyl or heteroaryl alkyloptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups, or, alternatively, R⁸ together with R⁷ mayform part of a benzo, naptho or polycyclic aryleno group which isoptionally substituted with one or more of the same or different R^(a)or suitable R^(b) groups; R¹¹ and R¹⁵ are each, independently of oneanother, selected from halo, (C1-C20) alkyl, haloalkyl, —OR^(y),—SR^(y), —SOR^(y), —SO₂R^(y), and nitrile; R¹², R¹³ and R¹⁴ are each,independently of one another, selected from hydrogen, R^(x), (C1-C20)alkyl or heteroalkyl optionally substituted with one or more of the sameor different R^(b) groups, (C5-C20) aryl or heteroaryl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups and (C6-C40) arylalkyl or heteroarylalkyl optionallysubstituted with one or more of the same or different R^(a) or suitableR^(b) groups; R^(x) is selected from —NR^(c)R^(c), —OR^(d), —SR^(d),halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, —N₃, —S(O)R^(d),—S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c), —S(O)₂NR^(c)R^(c),—OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d), —OS(O)₂NR^(c)R^(c),—C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c), —C(NH)NR^(c)R^(c),—OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and —OC(NH)NR^(c)R^(c);R^(y) is selected from (C1-C20) alkyls or heteroalkyls optionallysubstituted with lipophilic substituents, (C5-C20) aryls or heteroarylsoptionally substituted with lipophilic substituents and (C2-C26)arylalkyl or heteroarylalkyls optionally substituted with lipophilicsubstituents; R^(a) is selected from hydrogen, (C1-C8) alkyl orheteroalkyl, (C5-C20) aryl or heteroaryl and (C6-C28) arylalkyl orheteroarylalkyl; R^(b) is selected from —NR^(c)R^(c), ═O, —OR^(d), ═S,—SR^(d), ═NR^(d), ═NOR^(d), halo, haloalkyl, —CN, —NC, —OCN, —SCN, —NO,—NO₂, ═N₂, —N₃, —S(O)R^(d), —S(O)₂R^(d), —S(O)₂OR^(d), —S(O)NR^(c)R^(c),—S(O)₂NR^(c)R^(c), —OS(O)R^(d), —OS(O)₂R^(d), —OS(O)₂OR^(d),—OS(O)₂NR^(c)R^(c), —C(O)R^(d), —C(O)OR^(d), —C(O)NR^(c)R^(c),—C(NH)NR^(c)R^(c), —OC(O)R^(d), —OC(O)OR^(d), —OC(O)NR^(c)R^(c) and—OC(NH)NR^(c)R^(c); each R^(c) is independently hydrogen or R^(d), or,alternatively, each R^(c) is taken together with the nitrogen atom towhich it is bonded to form a 5 to 8-membered saturated or unsaturatedring which may optionally include one or more of the same or differentadditional heteroatoms and which may optionally be substituted with oneor more of the same or different R^(a) or R^(d) groups; each R^(d) isindependently R^(a) or R^(a) substituted with one or more of the same ordifferent R^(a) or R^(e) groups; and each R^(e) is selected from—NR^(a)R^(a), ═O, —OR^(a), ═S, —SR^(a), ═NR^(a), ═NOR^(a), halo,haloalkyl, —CN, —NC, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —S(O)R^(a),—S(O)₂R^(a), —S(O)₂OR^(a), —S(O)NR^(a)R^(a), —S(O)₂NR^(a)R^(a),—OS(O)R^(a), —OS(O)₂R^(a), —OS(O)₂OR^(a), —OS(O)₂NR^(a)R^(a),—C(O)R^(a), —C(O)OR^(a), —C(O)NR^(a)R^(a), —C(NH)NR^(a)R^(a),—OC(O)R^(a), OC(O)OR^(a), —OC(O)NR^(a)R^(a) and —OC(NH)NR^(a)R^(a), or,alternatively, one or more of R², R^(2′), R^(3′), R^(3″), R⁴, R^(4′),R⁵, R^(5′), R^(6′), R^(6″), R⁷, R^(7′), R¹², R¹³ and R¹⁴ are each,independently of one another, selected from the group consisting of—S-LGand —S¹-LK-S²—CS, wherein each S, which may be the same or different, isa direct bond or linker; each LG, which may be the same or different, isa linking group; each S¹ and S², independently, is a direct bond orlinker; each LK is a linkage formed from a reacted linking group, andeach CS, which may be the same or different is a conjugated substance.51. The phenyl xanthene dye of any one of claims 48, 49 and 50 whereinat least one of R¹¹ and R¹⁵ is selected from an alkoxy, halo, haloalkyland nitrile.
 52. The phenyl xanthene dye of any of claims 48, 49 and 50wherein both of R¹¹ and R¹⁵ are selected, independently, from an alkoxy,halo and haloalkyl groups.
 53. The phenyl xanthene dye of any one ofclaims 48, 49 and 50 wherein the R¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are,independently, selected from hydrogen, alkoxy, halo and haloalkylgroups.
 54. The phenyl xanthene dye of any one of claims 48, 49 and 50wherein R¹¹ and R¹⁵ are identical.
 55. The phenyl xanthene dye of anyone of claims 48, 49 and 50 wherein R¹¹ and R¹⁵ are identical and R¹²,R¹³, and R¹⁴ are, identically, selected from hydrogen or a substituentdifferent than R¹¹and R¹⁵.
 56. The phenyl xanthene dye of claim any oneof claims 48, 49 and 50 wherein R¹¹, R¹³ and R¹⁵ are identical and R¹²and R¹⁴ are, identically, selected from hydrogen or a substituentdifferent from R¹¹, R¹³ and R¹⁵.
 57. The phenyl xanthene dye of claimany one of claims 48, 49 and 50 wherein R¹¹, R¹², R¹⁴ and R¹⁵ are andR¹³ is hydrogen or a substituent different from R¹¹, R¹², R¹⁴ and R¹⁵.58. The phenyl xanthene dye of any one of claims 48, 49 and 50 whereinR¹¹, R¹², R¹³, R¹⁴ and R¹⁵ are identical.
 59. The phenyl xanthene dye ofany one of claims 48, 49, and 50 wherein A is a hydroxyl and B is anoxo.
 60. The phenyl xanthene dye of any one of claims 48, 49, and 50wherein A is an amine and B is an oxo.
 61. The phenyl xanthene dye ofany one of claims 48, 49 and 50 wherein A is an amine and B is animminium.
 62. The phenyl xanthene dye of any one of claims 48, 49, and50, wherein the dye is lipid soluble due to the presence of one or morelipophilic groups.
 63. The phenyl xanthene dye of any one of claim 62wherein the lipophilic groups are selected from (C4-C20) alkyls,(C5-C40) aryls, (C6-C40) arylalkyls.
 64. The phenyl xanthene dye of anyof claims 62 wherein said dye is a lipid soluble rhodamine in at leastone of the exocyclic amine and the exocyclic imminium nitrogens aresubstituted with one or more lipophilic groups.
 65. The phenyl xanthenedye of any one of claims 62 wherein said dye is a lipid soluble rhodolin which the exocyclic amine nitrogen is substituted with one or morelipophilic groups.
 66. The phenyl xanthene dye of any one of claims 48,49 and 50 wherein one of R², R^(2′), R⁴, R^(4′), R^(5′), R⁵, R^(7′), R⁷,R¹², R¹³, and R¹⁴ is selected from —S-LG and —S¹-LK-S²—SC.
 67. Thephenyl xanthene dye of any one of claims 48, 49 and 50 wherein one ofR², R^(2′), R⁴, R^(4′)R^(5′), R⁵, R^(7′), R⁷, R¹², R¹³, and R¹⁴is —S-LG.68. The phenyl xanthene dye of claim 67 wherein —S is a direct bond. 69.The phenyl xanthene dye of claim 67 in wherein —S is a spacing moiety.70. The phenyl xanthene of claim 69 wherein S is a spacing moietyselected from substituted and unsubstituted alkyldiyls,heteroalkyldiyls, aryldiyl and heteroaryldiyls.
 71. The phenyl xanthenedye of claim 67 wherein -LG is an electrophilic group selected fromactivated esters, acrylamides, acyl azides, acyl halides, acyl nitriles,aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, arylhalides, aziridines, boronates, carboxylic acids, carbodiimides,diazoalkenes, epoxides, haloacetamides, halotriazines, imido esters,isocyanates, isothiocyanates, maleimides, phosphoramidites, silylhalides, sulfonate esters, and sulfonyl halides.
 72. The phenyl xanthenedye of claim 67 wherein -LG is a nucleophilic group selected fromamines, anilines, alcohols, phenols, thiols, hydrazines, andhydroxylamines.
 73. The phenyl xanthene dye of any one of claims 48, 49and 50 wherein one of R², R^(2′), R⁴, R^(4′), R^(5′), R⁵, R^(7′), R⁷,R¹², R¹³, and R¹⁴ is —S¹-LK-S²—CS.
 74. The phenyl xanthene dye of claim73 wherein S¹ and S² are, independently, spacing moieties selected fromsubstituted and unsubstituted alkyldiyls, heteroalkyldiyls, aryldiyl andheteroaryldiyls.
 75. The phenyl xanthene dye of claim 73 wherein —CS isa polymer, glass or metal substrate.
 77. The phenyl xanthene dye ofclaim 73 wherein —CS is a biomolecule.
 78. The phenyl xanthene dye ofclaim 77 wherein the biological molecule is selected from peptides,polypeptides, proteins, enzymes, receptors, antibodies, hormones,polysaccharides, carbohydrates, oligonucleotides, polynucleotides andnucleic acids.
 79. The phenyl xanthene dye of claim 73, wherein —CS isan amino acid or a nucleoside.
 80. The phenyl xanthene dye of claim 73in which —CS is a second dye.
 81. The phenyl xanthene dye of claim 80 inwhich the second dye is an energy transfer donor or an energy transferacceptor.
 82. The phenyl xanthene dye of claim 80 in which the seconddye is selected from phenyl xanthene dyes, cyanine dyes, phthalocyaninedyes, squaraine dyes, acridine dyes, alizarene dyes, azo dyes,anthraquinine dyes, bodipy dyes, coumarin dyes, lanthanide complexes,oxazine dyes, phenazathionium dyes, phenazoxonium dyes, porphyorin dyes,pyrene dyes, pyrilium dyes, perylene dyes, phenoxazine dyes, andphenezine dyes.
 83. The phenyl xanthene dye of claim 80 in which thesecond dye is a phenyl xanthene dye.
 84. The phenyl xanthene dye ofclaim 80 in which the second dye is a phenyl xanthene that comprises oneof core structures (I), (II) and (III).